Cloning, sequencing and expression of a gene encoding an eukaryotic amino acid racemase, and diagnostic, therapeutic, and vaccination applications of parasite and viral mitogens

ABSTRACT

A method of preventing or inhibiting infection by a parasite or virus in vivo comprises administering to a human in need thereof a parasite or virus mitogen in a sub-mitogenic amount sufficient to induce a protective immune response against the parasite or virus in the human.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on and claims the benefit of each ofthe following applications: U.S. Provisional Application Ser. No.60/168,631, filed Dec. 3, 1999 (attorney docket no. 03495.6044); U.S.Provisional Application Ser. No. 60/220,207, filed Jul. 24, 2000(attorney docket no. 3495.6054); and U.S. Provisional Application Ser.No. 60/221,117, filed Jul. 27, 2000 (attorney docket no. 3495.6055). Theentire disclosure of each of these applications is relied upon andincorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] This invention relates to the discovery of a new gene, which isthe first isolated and cloned, that encodes an amino acid eukaryoticracemase. The invention covers, particularly, the Tc45 gene encoding aTrypanosoma cruzi-derived B-cell mitogen. The encoded protein also is aeukaryotic proline racemase. The invention also relates to a process ofproduction of D-amino acid using an eukaryotic amino acid racemase. Thisinvention also relates to the use of the protein encoded by the Tc45gene to induce a protective immune response against T. cruzi infectionin a human. This invention also relates to methods of using otherparasite mitogens and viral mitogens for inducing protective immunityagainst the corresponding parasitic or viral infections in humans.

[0003] The process of production of an D-amino acid by using a L-aminoacid source comprises the use of an eukaryotic amino acid racemasespecific for the amino acid of interest, the said racemase beingproduced from a recombinant expression system containing a vector havinga polynucleotide sequence encoding the said enzyme. In prokaryotichosts, the racemases are known to be implicated in the synthesis ofD-amino acids and/or in the metabolism of L-amino acids. Therefore, thepresence of free D-amino acids in tumors and in progressive autoimmuneand degenerative diseases suggests the biological importance ofeukaryotic amino acid racemases. It is well known that proteins orpeptides containing D-amino acids are resistant to proteolysis by hostenzymes. In addition, such proteins containing D-amino acids, at leastone D-amino acid residue, can display antibiotic or immunogenicproperties.

[0004] Isolation and characterization of molecules playing a key role inparasite metabolism, or in their interactions with the host immunedefense, are fundamental for the development of rational strategies forvaccination and therapy. Attempts to provide effective immunity toparasites are limited by poor specific immune responses to parasiteantigenic molecules in early phases of infection. Lymphocyte polyclonalactivation is a generalized mechanism of immune evasion amongstpathogens. Such “parasite evasion” owes, at least in part, to therelease of mitogenic or superantigenic moieties that inhibit hostspecific responses by triggering polyclonal, parasite non-specificlymphocyte activation. The resulting non-specific immune responses areassociated with immunosuppression and autoimmunity, as observed in humanand experimental infections by the protozoan parasite Trypanosoma cruzi,the etiological agent of Chagas disease²⁻⁶.

[0005] To date, there is no effective treatment or vaccine againstTrypanosoma cruzi infection and Chagas disease pathology. Attempts toisolate immunodominant protective epitopes have failed¹. Using a mousemodel of T. cruzi infection it has previously been shown that reducedlevels of polyclonal lymphocyte responses correlate with resistance toinfection and cardiopathy^(2, 7-9). As we have suggested, and has beendemonstrated by Arala-Chaves³¹ for Candida albicans infections,mitogenic moieties can be used as vaccination targets to induce specificneutralization of the mitogen, thus aborting the microorganism“strategy” to deviate immune responses into non-specific polyclonalactivation and immunosuppression. Understanding the mechanismsunderlying “non-specific” lymphocyte activation may open the way fortheir neutralization, and thus allow for effective immune responsesagainst infectious agents. There is a need in the art for a moleculethat could be an appropriate target for such attempts.

[0006] Furthermore, there is a growing interest in the biological roleof D-amino acids, either as free molecules or within polypeptide chainsin human brain, tumors, anti-microbial and neuropeptides, as well as in“protein fatigue”³², suggesting widespread biological implications.Research on D-amino acids in living organisms has been hampered by theirdifficult detection. However, recent purification of a serine racemasefrom mammalian brain³³ indicates conservation throughout evolution.There also exists a need in the art for racemases that are specific forknown compounds.

SUMMARY OF THE INVENTION

[0007] This invention aids in fulfilling these needs in the art. Moreparticularly, this invention relates to the characterization of aparasite molecule implicated in polyclonal responses that may serve as anovel target for vaccination and therapy. After identifying a proteinwith B-cell mitogenic properties in culture supernatants of infectiveparasite forms, the corresponding gene was cloned and its genomicorganization was characterized. The protein has been characterized as acofactor independent proline racemase, with strong homology to theproline racemase isolated from Clostridium sticklandii, thus providingthe first report on an eukaryotic amino acid racemase gene.

[0008] In particular, this invention provides a purified peptidecomprising an amino acid sequence (SEQ ID NOS: 1, 2, 3 and 4) encoded bythe Tc45 gene. This invention also provides polypeptide fragmentsderived from SEQ ID NOS: 7,8, 9, 10 and 11 containing at least 10 aminoacids.

[0009] This invention additionally provides purified polynucleotidescomprising the nucleic acid sequences of the Tc45 gene (SEQ ID NOS: 7,8, 9, 10 and 11). This invention also provides nucleic acid fragmentsderived from SEQ ID NOS: 7, 8, 9, 10 and 11 containing 15 to 40nucleotides.

[0010] Additionally, the invention includes a purified polynucleotidethat hybridizes specifically under conditions of moderate stringencywith a polynucleotide of SEQ ID NOS: 7,8, 9, and 10.

[0011] SEQ. ID 7 represents the full nucleotide sequence encoding theTrypanosoma cruzi proline racemase and N-terminal signal sequence andthe 5′ and 3′ flanking non-coding regions.

[0012] The SEQ ID 8 represents the full nucleotide sequence and itscorresponding polypeptide sequences [including the N terminal signalsequence and the 3′ non-coding flanking region] coding for a prolineracemase of T. cruzi.

[0013] The construct as disclosed in SEQ ID 8 deleted of the 3′non-coding flanking region and inserted in the PET28 vector (NOVAGEN),transformed in E. coli DH5 α was deposited at the CNCM under theaccession number I-2344.

[0014] A derived construct of I-2344 deleted of nucleotide sequencecorresponding to the signal peptide coding sequences, which is describedin SEQ ID 3, is used for the production of a recombinant active prolineracemase in E. coli. The E. coli DH 5α containing the plasmid with aninsert of 239 base pairs deposited at CNCM under accession numberI-2221, was obtained after amplification of the region by PCR techniquewith the primers SEQ ID Nos. 12 and 13. The insert was cloned into pTOPO II commercialized by INVITROGEN and then transformed in E. coli.

[0015] The invention further includes polynucleotide fragmentscomprising at least 10 nucleotides capable of hybridization underconditions of moderate stringency conditions with any one of thenucleotide sequences enumerated above.

[0016] In another embodiment of the invention, a recombinant DNAsequence comprising at least one nucleotide sequence enumerated aboveand under the control of regulatory elements that regulate theexpression of racemase activity in a host is provided.

[0017] The invention also includes a recombinant host cell comprising apolynucleotide sequence enumerated above or the recombinant vectordefined above.

[0018] In still a further embodiment of the invention, a method ofdetecting parasitic strains that contain the polynucleotide sequencesset forth above is provided.

[0019] Additionally, the invention includes kits for the detection ofthe presence of parasitic strains that contain the polynucleotidesequences set forth above.

[0020] The invention also contemplates antibodies recognizing peptidefragments or polypeptides encoded by the polynucleotide sequencesenumerated above.

[0021] Still further, the invention provides for a screening method foractive molecules for the treatment of infections due to parasites,particularly T. Cruzi, based on the detection of activity of thesemolecules on parasites.

[0022] This invention further provides an immunizing compositioncontaining at least a purified protein, or a fragment thereof, capableof inducing an immune response in vivo. The immune response can be amitogenic polyclonal immunoresponse in vivo. The immunizing compositionis suitable for use against a parasite infection under sub-mitogenicdoses.

[0023] This invention also provides a process to access the mitogenicityof a molecule called mitogen and the procedures to determine thesub-mitogenic dose suitable as an immunizing composition for use againsta parasite infection.

[0024] A vaccine composition of the invention for use against a T cruziinfection comprises a purified 38 to P45 kda protein or a fragmentthereof .

[0025] A method of inhibiting an eukaryotic protein with an amino acidracemase activity according to the invention comprises treating apatient by administering an effective amount of a molecule that inhibitsthe eukaryotic protein. The parasite can be T. cruzi.

[0026] This invention also provides a process for screening a moleculecapable of inhibiting the amino acid racemase activity of an eukaryoticprotein comprising the steps of:

[0027] contacting the purified eukaryotic racemase protein with standarddoses of a molecule to be tested;

[0028] measuring inhibition of racemase activity; and

[0029] selecting the molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] This invention will be more fully described with reference to thedrawings in which:

[0031]FIG. 1 depicts the proliferative activity of total spleen cellsstimulated in vitro by a 45 kDa B-cell polyclonal activator isolatedfrom parasite culture supernatants.

[0032] a. Proliferative activity of total spleen cells stimulated invitro by increasing concentrations (below graph) of total proteins fromculture supernatants of metacyclic trypomastigotes differentiated invitro. Inset, Proliferation of total (▪) or T cell-depleted (/)splenocytes in the presence of proteins from total culture supernatantc.p.m., counts per minute.

[0033] b. Proliferative activity of total spleen cells stimulated invitro by HPLC pooled fractions at 24 h (▪), 48 h (□) and 72 h (/).³H-thymidine uptake (c.p.m., counts per minute) after 48 h in thepresence of concavalin A and lipopolysaccharide was 32,000 and 3,500counts per minute, respectively. To rule out the possibility ofmitogenic effect due to lipopolysaccharide contamination in the samples,proliferation assays were also done using freshly recovered splenocytesfrom the ‘lipopolysaccharide non-responder’ mouse strain C3H/He; thesame levels of proliferation were obtained.

[0034] c. 8% SDS-PAGE analysis of fractions 21-24 (silver staining).Left margin, molecular size markers. Arrow: correspond to a 45 kDa band.

[0035]FIG. 2 shows the homology or similarity between the Tc45 protein(Tc) [SEQ ID NO:1] and the bacterial proline racemases Clostridiumsticklandii (Cs) [SEQ ID NO.:5] and Pseudomonas aeruginosa (Pa) [SEQ IDNO.:6]. The computer-predicted signal peptide is indicated by a doublearrow; peptide sequences obtained by microsequencing appear underlined;peptides used for designing degenerate primers appear in italiccharacters. Proline racemase active sites are boxed; dashes indicategaps generated for best fit.

[0036]FIG. 3 shows the genomic organization and transcription of theTc45 gene.

[0037] a. Southern blot analysis of 5 μg of T. cruzi genomic DNAdigested with indicated restriction enzymes and hybridized with a³²P-labeled probe, including the TcPA45 coding sequence, is shown.Molecular weights are indicated.

[0038] b. is a Northern blot analysis of 20 μg of total RNA fromepimastigotes forms hybridized with a ³²P-labeled probe as above.Molecular weights [Kb] are indicated.

[0039] c. mRNA expression of TcPA4S in different life stages of theparasite, shown by electrophoresis of gene fragments obtained byspecific reverse transcription from total parasite RNA followed by PCRamplification using the sequences of the mini-exon (spliced leader) andR-300-45 primers and subsequent amplification of a 170-bp internalfragment. M1, M2 and M3, molecular size markers (sizes, left and rightmargins). First-strand cDNA reactions were done in the presence (+) orabsence (−) of reverse transcriptase, to exclude the possibility offurther PCR amplification of fragments due to genomic DNA contamination,C1 and C2, internal negative (no template) controls; E, epimastigote, T,typomastigote and M, metacyllic.

[0040]FIG. 4 shows the results of characterization of rTcPA45 activitiesin vitro.

[0041] a. 8% SDS-PAGE gel of rTcPA45 (Coomasie blue staining).

[0042] b. Proliferative activity of total splenocytes (5×10⁴ cells/well)in the presence of increasing concentrations of rTcPA45 (μg/ml).

[0043] c. Percent racemisation of L-proline, D-proline,L-hydroxy-proline, D-hydroxy-proline substrates. Reaction conditions:0.2 M Na-acetate/25 mM β-mercaptoethanol buffer, pH 6, rTcPA45 (3μg/ml), 30 min incubation at 37° C. in 500 μl. The reaction was stoppedby incubating for 10 min at 80° C.

[0044] d. Percent inhibition of racemization of 80 mM L-proline in thepresence of several inhibitors.

[0045] e. Percent racemization of L-proline (80 mM) as a function of pH(buffers used were 0.2 M: Na-acetate, K-phosphate and Tris-HCl andcontained 25 mM β-mercaptoethanol).

[0046]FIG. 5 shows the nucleotide sequence [SEQ ID NO: 17] and peptidesequence [SEQ ID NO: 1] of TcPA45, The polypyrimidine rich region,splice leader acceptor sites, signal peptide, and polyadenylation siteare indicated. The peptide sequences obtained by microsequencing of thenative TcPA45 protein are underlined.

[0047]FIG. 6 is a demonstration of a cytosolic proline racemase inepimastigote forms of T. cruzi.

[0048] Western blot: membranes containing in:

[0049] Lane 1: total epimastigote extract from 5×10⁵ epimastigote forms

[0050] Lane 2: Epimastigote culture supernatant

[0051] Lane 3: Soluble fraction of epimastigote extract (cytosolic) from5×10⁵ epimastigote forms.

[0052] Lane 4: Insoluble fraction of epimastigote extract from 5×10⁵epimastigote forms

[0053] Lane 5: as in lane 1, from 10× more parasite forms (5×10⁶epimastigotes)

[0054] Lane 6: as in lane 3, from 10× more parasite forms (5×10⁶epimastigotes)

[0055] Lane 7: as in lane 2

[0056] Lane 8: as in lane 4, from 10× more parasite forms (5×10⁶epimastigotes)

[0057] Membranes were incubated with mouse polyclonal antibodies raisedagainst the rTcPA45 protein (primary antibody). Second step reaction wasdone with goat anti-mouse IgG-horseradish peroxidase (human absorbed).Reactivities were developed by ECL-chemiluminescence Amersham kit. FIG.6 represents 10 seconds exposure of the film.

[0058]FIG. 7 depicts the results of an ELISPOT assay. Spots correspondto immunoglobulin-producing B-cell (of a particular isotype) directed tothe coated antigen (here: goat anti-mouse immunoglobulins or rTcPA45).See also Example 10 and Table 1.

[0059]FIG. 8 depicts the results of DNA vaccination using various DNAconstructs. See also Example 11.

[0060]FIG. 9 Characterization of rTcPA45 mitogenic activity.

[0061] Proliferative activity of total splenocytes obtained from athymicor euthymic mice in the presence of rTcPA45 or lipopolysaccharide, at 24h (□), 48 h (/) or 72 h (▪).

[0062]FIG. 10 Differential expression of rTcPA45 protein in theparasite.

[0063] a. Cellular localization of the Tc45 protein in different lifestages of the parasite, shown by indirect immunofluorescence usingpolyclonal mouse serum against rTcPA45 followed by staining with theAlexa 488™ goat antibody against mouse IgG (H+L), F(ab′)₂ fragmentconjugate (bottom row), compared with control staining using the Alexa488™ F(ab′)₂ fragment conjugate alone (top left) or after incubation ofthe parasites with serum from chronically infected mice (Chronic serum).

[0064] b and c. Detection of Tc45 protein in total extracts ofepimastigote (E), metacyclic (M) and trypomastigote (T) forms of theparasite, compared with recombinant rTcPA45 (R) protein, by western blotanalysis. Arrows, calculated molecular weights for isoforms of theTcPA45 (R) protein.

[0065] d. Presence of the 39-kDa isoform of the Tc45 protein in total(Et), soluble (Ese) and insoluble (Emb) sonic extracts of non-infectiveepimastigote forms of the parasite, compared with its absence in culturesupernatants (Ecs). Molecular sizes (b-d), left margins.

[0066]FIG. 11 Correlation between mitogenic and racemase activities.

[0067] a. Proliferative activity of total mouse splenocytes in thepresence of rTcPA45 protein that is enzymatically active (rTcPA45) orlacking racemase activity by being heated (80°-rTcPA45), by long termstorage at 4° C. (Ina-rTcPA45), or by pre-incubation of rTcPA45 withpyrrole-Z carboxylic acid (rTcPA45+1 mM PCA), Iodoacetamide (rTcPA45+1mM IAA) or iodoacetate (rTcPA45+1 mM IAC) inhibitors, compared with 1 mMpyrrole-2-carboxylic acid 1 mMPCA) alone.

[0068] b. Competitive inhibition of rTcPA45-induced proliferativeactivity of total mouse lymphocytes by increasing concentrations of L-or D-proline substrates. Controls, cultures of splenocytes with 50 mM L-or D-proline alone. c.p.m., counts per minute.

[0069]FIG. 12 depicts the results of Western blot of mouse specificimmune responses directed to mitogenic rTcPA45 following immunization.See also Example 15.

[0070]FIG. 13 depicts two dose-response curves for two differenthypothetical mitogens, A and B, in an assay of proliferative activitydescribed hereinafter.

[0071]FIG. 14 depicts the results of immunization according to theinvention.

[0072] A. DNA vaccination. 8 week old BALB/c mice (5 mice/group) wereinjected 3 times (i.m., 50 μg of plasmid/femoral quadriceps) at 3 weeksinterval with the following constructs: pcDNA3 vector alone as control,or pcDNA3 containing the full encoding sequence of the TcPA45 gene with(Long) or without (Short) the fragment encoding the signal peptide. Micewere challenged 4 weeks after the last injection with 10⁴ infectiveforms of the parasite/mouse, and the parasitemia was scored during 35days.

[0073] OBS. It is worth noting that BALB/c mice were treated by almost 2months (9 weeks) to follow the vaccination protocol and were challengedat 21 weeks of age. Its is well known that mice of more than 9 weeks ofage are naturally more resistant to the experimental infection withTrypanosoma cruzi and no morality is observed.

[0074] The results using this vaccination protocol revealed that 3injections of pcDNA3 containing either the Short or Long encodingsequenvces of the TcPA45 gene, are able to reduce by more than 85% theparasitemia levels.

[0075] B. rTcPA45 injection. 6 week old BALB/c mice were injectedintraperitoneally (i.p.) with 10 ηg of rTcPA45 and then boosted i.p. oneweek later with 50 μg of rTcPA45. Mice were challenged 1 week after theboost with 10⁴ infective forms of the parasite/mouse, and theparasitemia was scored during 30 days. Control mice did not receive anytreatment before infection.

DETAILED DESCRIPTION

[0076] Having noted that proteins released from trypomastigote forms ofTrypanosoma cruzi behave as polyclonal B-cell activators¹⁰, it washypothesized that infective metacyclic forms would display an increasedproduction of mitogenic molecules, thereby promoting such a mechanism ofimmune evasion. To investigate the release of proteins with B-cellmitogenic activity by T. cruzi, culture supernatants of in vitrodifferentiated metacyclic trypornastigotes in a protein-free definedmedium were produced¹¹. Lymphocyte mitogenic activity in totalconcentrated culture supernatants was confirmed by lymphocyteproliferation assays.

[0077] Briefly, freshly recovered splenocytes from 8 week old maleBalb/c mice seeded at a concentration of 5×10⁴ cells/well were incubatedfor 24, 48 and 72 h in 5% FCS RPMI-1640 medium. A 16 h ³H-thymidinepulse at 1 μCi/well was performed before harvesting. ³H-Thymidine uptakewas determined in a beta-plate liquid scintillation counter(LKB-Wallac). All points were done in triplicate and the correspondingstandard deviation calculated. For comparison, ³H-thymidine uptake after48 h in the presence of ConA (10 μg/ml) and LPS (5 μg/ml) was 32000c.p.m. and 3500 c.p.m., respectively. T-cell depletion was performed byincubating freshly recovered spleen cells for 30 min at 37° C. in thepresence of anti-Thy 1.2 monoclonal antibody (Cedarlane) and rabbitcomplement (Cedarlane). In order to rule out a possible mitogenic effectdue to LPS contamination in samples, proliferation assays were alsoperformed on freshly recovered splenocytes from the LPS non-respondermouse strain C3H/Hej. Same levels of proliferation were verified. Balb/cmice were purchased from Charles River, France. C3H/Hej mice weremaintained in our animal facilities.

[0078] Proliferation is sustained over a 72 h period of culture in adose dependent manner as shown in FIG. 1a. Increasing concentrations (μgprotein/ml) of total culture supernatant of in vitro differentiatedmetacyclic trypomastigotes are shown in this FIG. The inset showsproliferation of total or T-cell depleted splenocytes in the presence of0.6 μg/ml of total culture supernatant. As for other B-cell mitogens,the same level of proliferation was observed when supernatants weretested on T-cell depleted splenocytes (FIG. 1a).

[0079] To identify the molecules responsible for mitogenic activity, theparasite proteins present in the metacyclic culture supernatants werefractionated by HPLC anion-exchanger chromatography, and the resultingfractions were tested as above. Fractions 22-24 (eluted at 368mM<NH₄-acetate<467 mM) showed the highest ³H-thymidine uptake at 24hours. The results are shown in FIG. 1b. 0.5 μg/ml of HPLC pooledfractions were resuspended in RPMI.

[0080] Previous observations using DEAE-chromatography-purified parasiteculture supernatants had shown that a protein fraction of 4045 kDa wasable to induce B-cell activation and proliferation in vivo. Theestimation of this range of molecular weight was done more or less 10%and compared with standard molecular weight kit markers commercializedby BioLabs (USA).

[0081] SDS-PAGE analysis of the HPLC fractions revealed the presence ofa protein with an apparent molecular weight around 45 kDa only infractions 21-24 as shown in FIG. 1c. (8% SDS-PAGE gel of fractions 21 to24 (silver staining)). The isoelectric point of the protein wasestimated between 4.5 and 5.0 by isoelectric focusing. These dataindicated that a protein with B-cell mitogenic activity was present infractions 21-24 eluted from the anionic matrix, and the 45 kDa proteinband was thus selected as the main candidate.

[0082] To obtain peptide sequences from the 45 kDa protein, which wasidentified as Tc45, and allow for subsequent PCR-assisted cloning of agene fragment, the 45 kDa protein band was isolated by SDS-PAGE, and itsinternal digestion and microsequencing were undertaken. Six peptideswere isolated, sequenced, and were shown to have the followingsequences: (W)⁴³IIk⁴⁶ [SEQ ID NO:18]; I⁹⁰VTGSLP(D)I(S)G¹⁰⁰ [SEQ IDNO:19]; A¹⁸³TNVPWLDTPAGLVR¹⁹⁸ [SEQ ID NO:20]; V²⁴¹DIAFGGNF²⁴⁹ [SEQ IDNO:21]; N³¹⁶WIFGNR³²³ [SEQ ID NO:22], and M³³⁸ATLYAK³⁴⁴ [SEQ ID NO:23].These sequences are underlined in FIG. 2.

[0083] Peptides 4 [SEQ ID NO:21] and 5 [SEQ ID NO:22] were selected fordegenerate primer design on the basis of the relatively low level ofdegeneracy in their corresponding coding sequences. The sequences ofthese peptides are identified by italic characters in FIG. 2.

[0084] Reverse transcription was performed on total RNA from T. cruzitrypomastigote forms using reverse degenerate primers for both peptides.The resulting cDNA was then used as a template for PCR amplification.From all possible combinations, when using in the PCR reaction forwardprimer for peptide 4 (SEQ ID No.13), reverse primer for peptide 5, andtemplate cDNA synthesized with reverse primer for peptide 5 (SEQ IDNo.12), only one PCR product of 239 bp was shown to contain both primersafter cloning and sequencing. The sequence analysis revealed that thefragment contained an unique open reading frame (ORF) flanked bypeptides 4 [SEQ ID NO:21] and 5 [SEQ ID NO:22] coded in frame.

[0085] To obtain the full sequence of the Tc45 gene, the ³²P-labelled239 bp PCR product was used as a probe to screen a Trypanosoma cruziclone CL-Brener lambda Fix II genomic library. Four independent positivephages were isolated. Restriction analysis and Southern blothybridization revealed two types of patterns, each represented by twophages, suggesting that the Tc45 gene is present in at least two copiesper haploid genome.

[0086] The complete sequence of the Tc45 gene and flanking regions(GenBank accession no. AF195522), revealed an ORF of 423 codonscontaining all sequenced peptides (FIG. 2) [SEQ ID NO:1]. Computeranalysis predicts a 29 amino acid signal peptide [SEQ ID NO: 3] (doublearrowed in FIG. 2) suggesting active secretion by T cruzi. This is inagreement with the fact that the protein was purified from culturesupernatants. A poly-pyrimidine rich region and probable trans-spliceacceptor site is observed 56 and 7 base pairs (bp) upstream of the ATGcodon, respectively. Interestingly, an alternative trans-splicing signalis present about 170 bp upstream of the second ATG codon within thecoding region, which if used would allow the expression of a truncated,non-secreted protein lacking 69 amino acids, which is SEQ ID No. 4.Polyadenylation may take place at positions 1442 or 1443, which arepreceded by repeats of the triplet UUA, a motif found at the 3′ end ofother T. cruzi genes^(12, 13).

[0087] To investigate the genomic organization and transcription of theTc45 gene, Southern and Northern blot analyses were done. FIG. 4 depictsthe results of characterization of rTcPA45 activities. FIG. 4, part a,is an 8% SDS-PAGE gel of rTcPA45 (Coomasie blue staining). Part bdepicts the proliferative activity of total splenocytes (5×10⁴cells/well) in the presence of increasing concentrations of rTcPA45(μg/ml). Part c shows percent racemization of L-proline, D-proline,L-hydroxy-proline, D-hydroxy-proline substrates. Reaction conditionswere: 0.2 M Na-acetate/25 mM β-mercaptoethanol buffer, pH 6, rTcPA45 (3μg/ml), 30 min incubation at 37° C. in 500 μl. The reaction was stoppedby incubating for 10 min at 80° C. FIG. 4, part d, shows percentinhibition of racemization of 80 mM L-proline in the presence of severalinhibitors. Part e shows percent racemization of L-proline (80 mM) as afunction of pH (buffers used were 0.2 M: Na-acetate, K-phosphate andTris-HCl and contained 25 mM β-mercaptoethanol). Reactions were carriedout for 30 min at 37° C. and stopped for 5 min at 80° C. All reagentsand inhibitors were purchased from Sigma. The shift in optical rotationwas measured in a Polarimeter 241 MC (Perkin Elmer).

[0088] To investigate the genomic organization and transcription of theTc45 gene, we used Southern blot analysis; this indicated the presenceof two gene copies per haploid genome. There are probably two homologousTc45 genes (FIG. 3a). Digestion with BamHI and BglII producted twohybridizing bands, consistent with the presence of two gene copies, asthe probe has neither enzyme restriction site. High-molecular-weight DNAhybridized after probable partial digestion with SalI, consistent withthe absence of this site within the coding sequence covered by theprobe. Both PstI and TaqI cleaved within the probe and produced morethan one hybridizing band per gene copy. Preliminary results indicatedthat they are located on different chromosomes (data not shown) Northernblot analysis on total RNA from epimastigotes showed a transcript ofaround 1.5 kb, as expected from the genomic sequence (FIG. 3b). Weconfirmed the presence of the Tc45 mRNA in different parasite forms byreverse transcription PCR using primers specific for the Tc45 gene (FIG.3c). Several point mutations were already identified in the sequence ofthe putative Tc45-B gene copy representative of the second phage type(FIG. 2), and further transcriptional and functional analyses of thealleles are underway.

[0089] To identify homologies with other genes, we compared the Tc45-Agene copy and protein sequence to several databases. There was homology(nucleotide sequence, 57.7%; IDENTITY amino-acid sequence, 52.4%IDENTITY) with the only proline racemase described¹⁴, an intracellularhomodimetric protein isolated from Clostridum sticklandii. This enzymecatalyzes the interconversion between the L- and D-proline enantiomers,and its reaction mechanism has been studied extensively¹⁴. There wasalso homology with the translation of an ORF sequence from Pseudomonasaeruginosa in contig 53 of the unfinished Pseudomonas genome project(amino-acid sequence, 37.9% IDENTITY; C. sticklandii proline racemaseand the translation of the P. aeruginosa ORF, 47% IDENTITY. The C.sticklandii proline racemase active site has been identified¹⁵ and isconserved in the Tc45 protein. Homology with both bacterial proteinsstarted at amino acid 70 of Tc45 (FIG. 2). The presence of the KIIKpeptide (FIG. 2, underlining) in the Tc45 protein purified from parasiteculture supernatants confirmed the presence of the extra N-terminalportion of the protein released by T. cruzi metacyclic forms.

[0090] More particularly, the SDS-PAGE analysis of the over-expressedand purified protein is shown in FIG. 4a. Using in vitro proliferationassays of naive murine spleen cells, recombinant protein rTc45 was shownto display a similar mitogenic activity to the one observed with thenative protein fraction, purified from culture supernatants. Thus,rTcPA45 (for T. cruzi polyclonal activator 45) induces spleen lymphocyteproliferation, which increases with time over a 72 h period of culture(FIG. 4b). Proliferation is dose dependent, with a bell-shaped responsecurve (starting from 0.8 μg/ml, and peaking at 50 μg/ml) typical of allmitogens described to date. rTcPA45 is indeed a T cell-independentpolyclonal activator of B lymphocytes, as shown by the magnitude andincrease of the proliferative response of total spleen lymphocytesobtained from athymic mice compared with the response of lymphocytesfrom euthymic individuals (FIG. 9). Injection of 50 μg rTcPA45 in vivoinduced a 2 fold increase in spleen cell numbers by day 4, accompaniedby an increase in numbers of immunoglobulin (lg)-secreting B cells ofthe IgM, IgG2a, IgG2b and IgG3 isotypes (2.5 fold to 100×), whileshowing a complete lack of rTcPA45-specific Ig-secreting B cells,indicating the polyclonal B-cell mitogenicity of the protein (data notshown).

[0091] Injection of 50 μg of rTcPA45 in vivo induces a 2-fold increasein spleen cell numbers by day 7 accompanied by 2.5 to 4 fold increase innumbers of Ig-secreting B cells of IgM, IgG2a, and IgG2b isotypes, whileshowing a complete lack of Ig-secreting B cells directed to TcPA45demonstrating the polyclonal B-cell mitogenicity of the protein as shownin Example 10, Table I.

[0092] To confirm that the TcPA45 protein is indeed a proline racemase,in vitro biochemical assays were performed to measure the shift inoptical rotation of either L- or D-proline substrates. As can be seen inFIG. 4c, rTcPA45 racemises both L- and D-proline, but not L- orD-hydroxy-proline, nor any other natural L-amino acids. Such rTcPA45racemase activity is co-factor independent, notably of pyridoxalphosphate, and thus closely resembles the C. sticklandii prolineracemase^(I4).

[0093] Furthermore, the rTcPA45 enzymatic activity is inhibited todifferent extents by the presence of previously described inhibitors ⁴,such as maleic acid, iodoacetamide, iodoacetate, andpyrrole-2-carboxylic acid (FIG. 4d). Interestingly, rTcPA45 prolineracemase activity is maximal at pH 6 (FIG. 4e), two units lower thanthat of the bacterial enzyme^(I4). The optimal temperature for enzymaticactivity is 37° C., and the enzyme is inactivated by 10 min heating at80° C. (data not shown).

[0094] To analyze the cellular localization of the parasite TcPA45, weused immunofluorescence experiments with a polycyclonal serum raisedagainst rTcPA45. Whereas serum from chronically infected mice stainedtypomastigote cells uniformly, rTCPA45 specific antibodies stainedmostly the cytoplasm of epimastigote forms but not the nucleus or thekinetoplast (FIG. 10a). In vitro-differentiated metacyclic forms showeda less in tense and more diffuse pattern of cytoplasmic staining thandid epimastigotes. However, bloodstream trypomastigote forms werestrongly labeled at the flageliar pocket and the anterior and posteriorends of the parasite, and lightly along the flagellum and cytoplasm.These experiments substantiate the hypothesis that T. cruzi has anintracellular form of the proline racemase and the secretion might onlytake place in the infective forms. Western blot analysis of cellextracts of the parasite confirmed that the TcPA45 protein was presentin different developmental stages (FIG. 10b). We detected a TcPA45protein around 39 kDa in molecular mass in epimastigotes (non-infectiveinsect forms) and 41.S kDa in infective metacyclic trypomastigotes,compared with the computer-predicated molecular masses of 43.4 kDa and38 kDa, respectively, for a secreted and a non-secreted form of theprotein (FIG. 10c). Western blot analysis of the non-infectiveepimastigote cell stage showed Tc45 proline racemase mostly in thesoluble cellular fraction, only weakly in the cellular insolublefraction and absent from culture medium (FIG. 10d).

[0095] To confirm the relationship, if any, between the enzymatic andthe mitogenic activities of rTcPA45, we used in vitro proliferationassays with active rTcPA45 and different forms of the inactivatedenzyme. Unexpectedly, mitogenic activity was abolished when rTcPA45 wasinactivated by being heated or by long storage at 4° C., or wheneverenzymatic inhibition was achieved by pre-incubation of the protein withspecific (pyrrole-2-carboxylic acid) or nonspecific (iodoacetamide andiodoacetate) inhibitors of proline racemase (FIG. 11a). Mitogenicactivity was also affected considerably by supplementation of thecultures with increasing amounts of L- or D-proline, in a dose-dependentmanner, indicating that competitive inhibition occurred in the presenceof specific substrates (FIG. 11b). There was no cell proliferation whenlymphocytes were cultured in the presence of 50 mM L- or D-proline alone(FIG. 11b). Furthermore, mitogenic activity due to another B-cellmitogen was unaffected by the inhibitors or substances (data not shown).Although the antibodies against TcPA45 raised against the recombinantprotein were not able to inhibit racemization or to neutralize mitogenicactivity in vitro, and thus cannot be used to support a link betweenthese activities, the results indicate that a free and intact activesite of the rTcPA45 protein is necessary to allow mitogenicity.

[0096] This is the first description of an amino acid racemase gene inan eukaryotic organism. Thus, this invention relates to the biochemicalisolation, cloning, and molecular characterization of a B-cell mitogenreleased by Trypanosoma cruzi. Unexpectedly, this is also the firstdescription of a racemase in a parasite.

[0097] In bacteria, amino acid racemases are cytoplasmic proteinsparticipating in metabolic processes or in the synthesis ofpost-translationally modified peptides¹⁶. It is known that T. cruzi canuse L-proline as a major carbon source¹⁷, possibly through a D-prolineintermediate¹⁸. In this case, however, one might expect to find acytosolic proline racemase. Indeed, we have identified a cytosolicproline racemase in epimastigote forms of T. cruzi as shown in FIGS. 6and 10(b). This racemase presents a molecular weight of 38 kd in 10% SDSPage by using a standard molecular weight kit commercialized by BioLABS(USA). The estimation of the molecular weight is done more or less 10%around 38 kda. It is well established that proteins bearing D-aminoacids are highly resistant to eukaryotic proteases. Thus, it might alsobe possible that the parasite uses the racemization mechanism duringmetacyclogenesis to synthesize and express, on its surface, proteinscontaining D-proline, therefore ensuring a certain degree of resistanceto host-induced proteolytic mechanisms during cell invasion.Interestingly, T. cruzi differentiation from epimastigote totrypomastigote is induced in the presence of L-proline at pH 6 in theinsect's gut¹⁹ as well as during in vitro metacyclogenesis¹¹, and theproline racemase activity of the TcPA45 protein might be involved inthis process.

[0098]FIG. 2 shows homology between the Tc45 protein (Tc) and thebacterial proline racemases Clostridium sticklandii (Cs) and Pseudomonasaeruginosa (Pa). Identical residues appear in bold characters; thecomputer-predicted signal peptide is indicated by a double arrow;peptide sequences obtained by microsequencing appear underlined;peptides used for designing degenerate primers appear in italiccharacters. Proline racemase active sites are boxed; dashes indicategaps generated for best fit.

[0099] The homology of TcPA45 protein sequence with the Clostridiumsticklandii proline racemase starts at amino acid 70 (second methioninein the ORF of TcPA45 gene) allowing the speculation that T. cruzi hasacquired an extra 70 amino acid sequence N-terminal to the ancestralproline racemase (comprising a signal peptide for secretion) gaining theability to produce either a cytosolic or a secreted form of the proteinby differential trans-splicing of the mRNA. In addition, this processmight be differentially regulated at distinct developmental stages ofthe parasite. In this context, it could then be hypothesised thatepimastigote forms produce a cytosolic protein, while infectivetrypomastigotes secrete the same protein that activates B-cellpolyclonal responses.

[0100] Interestingly, the disruption of alanine racemase and the D-aminoacid aminotransferase genes of Listeria monocytogenes results in theinability of the bacteria to grow within the eukaryotic host cells²⁴.Both these gene products are involved in the synthesis of D-alanine thatis required for the production of a mucopeptide component of the cellwalls of virtually all bacteria. It remains to be investigated whetheror not such bacterial molecules are B-cell mitogens. Accordingly,working with random amino acid polymers, Sela and co-workers haveestablished that multichain polypeptides composed of D-amino acidsinduce antibody responses in a T-cell independent manner^(25, 26), aproperty that can be interpreted as equivalent to B-cellmitogenicity^(27, 28). Resistance of such D-amino acid peptides todegradation by host enzymes²⁹ could also explain the persistence ofpolyclonal responses (and immunosuppression)³⁰, even if parasiteproduction of the mitogen would be transient at the start of infection.

[0101] Thus, novel polynucleotides corresponding to the Tc45 gene fromCL strain (representative of lineage T. cruzi II) T. Cruzi have beenisolated and sequenced. The presence of the Tc45 gene has also beendemonstrated in a representative strain of another major lineage of T.cruzi [strain DM 28c, lineage T. cruzi I]. For a review onrecommendations on T. cruzi strain nomenclature see: Mem. Institut. OSW.Cruz, Vol. 94, Suppl. 1, page 429-432, 1999. These polynucleotidesinclude SEQ ID NOS: 7, 8, 9, 10, and 11. By “polynucleotides” accordingto the invention is meant the sequences referred to as SEQ ID NOS: 7, 8,9, 10, and 11, and the complementary sequences and/or the sequences ofpolynucleotides that hybridize to the referred sequences underconditions of moderate stringency. The moderate stringency conditionsare defined as washing conditions in 2× SSC at 55° C., and hybridizationoperated in 5× SSC at 55° C.

[0102] By “active molecule” according to the invention is meant amolecule capable of inhibiting the activity of the purified recombinantor native polypeptide as defined in the present invention.

[0103] Thus, the polynucleotides of SEQ ID NO: 7 and its fragments canbe used as probes or to select nucleotide primers notably for anamplification reaction, such as the amplification reactions furtherdescribed. PCR is described in the U.S. Pat. No. 4,683,202 granted toCetus Corp. The amplified fragments may be identified by agarose orpolyacrylamide gel electrophoresis, or by a capillary electrophoresis,or alternatively by a chromatography technique (gel filtration,hydrophobic chromatography, or ion exchange chromatography). Thespecificity of the amplification can be ensured by a molecularhybridization using as nucleic acid probes the polynucleotides derivedfrom SEQ ID NO: 7 and its fragments, oligonucleotides that arecomplementary to these polynucleotides or fragments thereof, or theiramplification products themselves.

[0104] Amplified nucleotide fragments are useful as probes inhybridization reactions in order to detect the presence of onepolynucleotide according to the present invention or in order to detectthe presence of a parasite of T. Cruzi strain carrying genes encodingracemase activity, in a biological sample. This invention also providesthe amplified nucleic acid fragments (“amplicons”) defined herein above.These probes and amplicons can be radioactively or non-radioactivelylabeled, using for example enzymes or fluorescent compounds.

[0105] Preferred nucleic acid fragments that can serve as primersaccording to the present invention are the following:5′TTICCRAADATIACIACGTT3′ [SEQ ID NO: 12] 5′ATHGCITTYGGIGGIAAYTTT3′ [SEQID NO: 13] 5′TTICCRAADATIACIACGTT3′ [SEQ ID NO: 14]5′CTCTCCCATGGGGCAGGAAAAGCTTCTG3′ [SEQ ID NO: 15]5′CTGAGCTCGACCAGATCTATCTGC3′ [SEQ ID NO: 16].

[0106] The primers can also be used as oligonucleotide probes tospecifically detect a polynucleotide according to the invention.

[0107] Other techniques related to nucleic acid amplification can alsobe used alternatively to the PCR technique. The Strand DisplacementAmplification (SDA) technique (Walker et al., 1992) is an isothermalamplification technique based on the ability of a restriction enzyme tocleave one of the strands at a recognition site (which is under ahemiphosphorothioate form), and on the property of a DNA polymerase toinitiate the synthesis of a new strand from the 3′ OH end generated bythe restriction enzyme, and on the property of this DNA polymerase todisplace the previously synthesized strand being localized downstream.

[0108] The SDA amplification technique is more easily performed than PCR(a single thermostated water bath device is necessary), and is fasterthan the other amplification methods. Thus, the present invention alsocomprises using the nucleic acid fragments according to the invention(primers) in a method of DNA or RNA amplification, such as the SDAtechnique. The polynucleotides of SEQ ID NO: 7 and its fragments,especially the primers according to the invention, are useful astechnical means for performing different target nucleic acidamplification methods, such as:

[0109] TAS (Transcription-based Amplification System), described by Kwohet al. in 1989;

[0110] SR (Self-Sustained Sequence Replication), described by Guatelliet al. in 1990;

[0111] NASBA (Nucleic acid Sequence Based Amplification), described byKievitis et al. in 1991; and

[0112] TMA (Transcription Mediated Amplification).

[0113] The polynucleotides of SEQ ID NO: 7 and its fragments, especiallythe primers according to the invention, are also useful as technicalmeans for performing methods for amplification or modification of anucleic acid used as a probe, such as:

[0114] LCR (Ligase Chain Reaction), described by Landegren et al. in1988 and improved by Barany et al. in 1991, who employ a thermostableligase;

[0115] RCR (Repair Chain Reaction), described by Segev et al. in 1992;

[0116] CPR (Cycling Probe Reaction), described by Duck et al. in 1990;and

[0117] Q-beta replicase reaction, described by Miele et al. in 1983 andimproved by Chu et al. in 1986, Lizardi et al. in 1988, and by Burg etal. and Stone et al. in 1996.

[0118] When the target polynucleotide to be detected is RNA, for examplemRNA, a reverse transcriptase enzyme can be used before theamplification reaction in order to obtain a cDNA from the RNA containedin the biological sample. The generated cDNA can be subsequently used asthe nucleic acid target for the primers or the probes used in anamplification process or a detection process according to the presentinvention.

[0119] Nucleic acid probes according to the present invention arespecific to detect a polynucleotide of the invention. By “specificprobes” according to the invention is meant any oligonucleotide thathybridizes with the polynucleotide of SEQ ID NO: 7, and which does nothybridize with unrelated sequences. Preferred oligonucleotide probesaccording to the invention are SEQ ID NOS:12, 13,14, 15, and 16.

[0120] In a specific embodiment, the purified polynucleotides accordingto the present invention encompass polynucleotides having at least 80%identity in their nucleic acid sequences with polynucleotide of SEQ IDNO: 7 or fragments thereof. By percentage of nucleotide identityaccording to the present invention is intended a percentage of identitybetween the corresponding bases of two homologous polynucleotides, thispercentage of identity being purely statistical and the differencesbetween two homologous polynucleotides being located at random and onthe whole length of said polynucleotides. The calculation was madeaccording to the software GCG and the program “gap.”

[0121] The oligonucleotide probes according to the present inventionhybridize specifically with a DNA or RNA molecule comprising all or partof the polynucleotide of SEQ ID NO: 7 under stringent conditions. As anillustrative embodiment, the stringent hybridization conditions used inorder to specifically detect a polynucleotide according to the presentinvention are advantageously the following:

[0122] Prehybridization and hybridization are performed as follows inorder to increase the probability for heterologous hybridization:

[0123] The prehybridization and hybridization are done at 50° C. in asolution containing 5× SSC and 1× Denhardt's solution.

[0124] The washings are performed as follows:

[0125] 2× SSC at 60° C. 3 times during 20 minutes each.

[0126] The non-labeled polynucleotides or oligonucleotides of theinvention can be directly used as probes. Nevertheless, thepolynucleotides or oligonucleotides are generally labeled with aradioactive element (³²P, ³⁵S, ³H, ¹²⁵I) or by a non-isotopic molecule(for example, biotin, acetylaminofluorene, digoxigenin,5-bromodesoxyuridin, fluorescein) in order to generate probes that areuseful for numerous applications. Examples of non-radioactive labelingof nucleic acid fragments are described in the French Patent No. FR 7810975 or by Urdea et al. or Sanchez-Pescador et al. 1988.

[0127] Other labeling techniques can also be used, such as thosedescribed in the French patents 2 422 956 and 2 518 755. Thehybridization step may be performed in different ways (Matthews et al.1988). A general method comprises immobilizing the nucleic acid that hasbeen extracted from the biological sample on a substrate(nitrocellulose, nylon, polystyrene) and then incubating, in definedconditions, the target nucleic acid with the probe. Subsequent to thehybridization step, the excess amount of the specific probe isdiscarded, and the hybrid molecules formed are detected by anappropriate method (radioactivity, fluorescence, or enzyme activitymeasurement).

[0128] Advantageously, the probes according to the present invention canhave structural characteristics such that they allow signalamplification, such structural characteristics being, for example,branched DNA probes as those described by Urdea et al. in 1991 or in theEuropean Patent No. 0 225 807 (Chiron).

[0129] In another advantageous embodiment of the present invention, theprobes described herein can be used as “capture probes”, and are forthis purpose immobilized on a substrate in order to capture the targetnucleic acid contained in a biological sample. The captured targetnucleic acid is subsequently detected with a second probe, whichrecognizes a sequence of the target nucleic acid that is different fromthe sequence recognized by the capture probe.

[0130] The oligonucleotide fragments useful as probes or primersaccording to the present invention can be prepared by cleavage of thepolynucleotide of SEQ ID NO: 7 by restriction enzymes, as described inSambrook et al. in 1989. Another appropriate preparation process of thenucleic acids of the invention containing at most 200 nucleotides (or200 bp if these molecules are double-stranded) comprises the followingsteps:

[0131] Synthesizing DNA using the automated methods, such asbeta-cyanethylphosphoramidite described in 1986;

[0132] cloning the thus obtained nucleic acids in an appropriate vector;and

[0133] purifying the nucleic acid by hybridizing to an appropriate probeaccording to the present invention.

[0134] A chemical method for producing the nucleic acids according tothe invention, which have a length of more than 200 nucleotides (or 200bp if these molecules are double-stranded), comprises the followingsteps:

[0135] Assembling the chemically synthesized oligonucleotides, which canhave different restriction sites at each end;

[0136] cloning the thus obtained nucleic acids in an appropriate vector;and

[0137] purifying the nucleic acid by hybridizing to an appropriate probeaccording to the present invention.

[0138] The oligonucleotide probes according to the present invention canalso be used in a detection device comprising a matrix library of probesimmobilized on a substrate, the sequence of each probe of a given lengthbeing localized in a shift of one or several bases, one from the other,each probe of the matrix library thus being complementary to a distinctsequence of the target nucleic acid. Optionally, the substrate of thematrix can be a material able to act as an electron donor, the detectionof the matrix positions in which hybridization has occurred beingsubsequently determined by an electronic device. Such matrix librariesof probes and methods of specific detection of a target nucleic acid aredescribed in European patent application No. 0 713 016, or PCTApplication No. WO 95 33846, or also PCT Application No. WO 95 11995(Affymax Technologies), PCT Application No. WO 97 02357 (AffymetrixInc.), and also in U.S. Pat. No. 5,202,231 (Drmanac), said patents andpatent applications being herein incorporated by reference.

[0139] The present invention also pertains to a family of recombinantplasmids containing at least a nucleic acid according to the invention.According to an advantageous embodiment, a recombinant plasmid comprisesa polynucleotide of SEQ ID NO: 7 or nucleic acid fragment thereof. Morespecifically, the following plasmids are part of the invention:

[0140] DH5[alpha]-pTc45 MIT (1335 bp) and

[0141] Dh5[α]-pTc45 MIT (239 bp).

[0142] A suitable vector for the expression in bacteria, and inparticular in E. coli, is pET-28 (Novagen), which allows the productionof a recombinant protein containing a 6× His affinity tag. The 6× Histag is placed at the C-terminus or N-terminus of the recombinantpolypeptide. The purified racemase is obtained by expression in E. colitransformed with pET-28 containing the insert of the plasmidcorresponding to the CNCM No. I-2344 deleted of the sequence of thesignal peptide as shown on SEQ ID NO: 9 and including the six C terminalhistidine residues. The expression of pET-28 with the insert was inducedby IPTG (1 millimolar) overnight at 20° C. resulting in a solublerecombinant racemase according to the invention. This racemase has amolecular weight of 45 kda in 8% SDS PAGE gel (see FIG. 4a) comparedwith standard molecular weight kit markers (BioLABS). After lysis of thebacterial cells by a French Press, followed by centrifugation (2000 gduring 15 minutes), the recombinant protein was purified from thesupernatant using nickel IMAC chromatography. (commercialized byPHARMACIA) and eluted in 0.5 molar imidazol buffer. The yield is between10 to 40 mg of protein for one liter of bacterial culture at 1.ODdensity. The OD at the beginning of the induction of the expression inthe recombinant bacterial was comprised between 0.6 to 1 OD. In theculture conditions as disclosed above, the majority of the recombinantprotein produced is in a soluble form and they are not favorable for theexpression of the proteins of the bacterial host. The estimation of themolecular weight of the purified recombinant protein is done more orless 10% around 45 kda.

[0143] The polypeptides according to the invention can also be preparedby conventional methods of chemical synthesis, either in a homogenoussolution or in solid phase. As an illustrative embodiment of suchchemical polypeptide synthesis techniques, the homogenous solutiontechnique described by Houbenweyl in 1974 may be cited.

[0144] The polypeptides of the invention are useful for the preparationof polyclonal or monoclonal antibodies that recognize the polypeptides(SEQ ID NOS: 1, 2, 3, and 4) or fragments thereof. The monoclonalantibodies can be prepared from hybridomas according to the techniquedescribed by Kohler and Milstein in 1975. The polyclonal antibodies canbe prepared by immunization of a mammal, especially a mouse or a rabbit,with a polypeptide according to the invention, which is combined with anadjuvant, and then by purifying specific antibodies contained in theserum of the immunized animal on a affinity chromatography column onwhich has previously been immobilized the polypeptide that has been usedas the antigen.

[0145] Consequently, the invention is also directed to a method fordetecting specifically the presence of a polypeptide according to theinvention in a biological sample. The method comprises:

[0146] a) bringing into contact the biological sample with an antibodyaccording to the invention; and

[0147] b) detecting antigen-antibody complex formed.

[0148] Also part of the invention is a diagnostic kit for in vitrodetecting the presence of a polypeptide according to the presentinvention in a biological sample. The kit comprises:

[0149] a polyclonal or monoclonal antibody as described above,optionally labeled; and

[0150] a reagent allowing the detection of the antigen-antibodycomplexes formed, wherein the reagent carries optionally a label, orbeing able to be recognized itself by a labeled reagent, moreparticularly in the case when the above-mentioned monoclonal orpolyclonal antibody is not labeled by itself.

[0151] Indeed, the monoclonal or polyclonal antibodies according to thepresent invention are useful as detection means in order to identify orcharacterize a T. Cruzi strain carrying TC45 genes.

[0152] The invention also pertains to:

[0153] A purified polypeptide or a peptide fragment having at least 10amino acids, which is recognized by antibodies directed against apolynucleotide or peptide sequence according to the invention.

[0154] A polynucleotide comprising the full length coding sequence ofthe Tc45 gene sequence according to the invention.

[0155] A monoclonal or polyclonal antibody directed against apolypeptide or a peptide fragment encoded by the polynucleotidesequences according to the invention.

[0156] A method of detecting the presence of parasite harboring thepolynucleotide sequences according to the invention in a biologicalsample comprising:

[0157] a) contacting DNA or RNA of the biological sample with a primeror a probe according to the invention, which hybridizes with anucleotide sequence;

[0158] b) amplifying the nucleotide sequence using said primer or saidprobe; and

[0159] c) detecting the hybridized complex formed between said primer orprobe with the DNA or RNA.

[0160] A kit for detecting the presence of a parasite harboring thepolynucleotide sequences according to the invention in a biologicalsample, comprises:

[0161] a) a polynucleotide probe according to the invention; and

[0162] b) reagents necessary to perform a nucleic acid hybridizationreaction.

[0163] A method of screening active molecules for the treatment of theinfections due to a parasite, comprises the steps of:

[0164] a) bringing into contact a parasite containing the polynucleotidesequences according to the invention with the molecule; and

[0165] b) measuring an activity of the active molecule on the parasite.

[0166] An in vitro method of screening for an active molecule capable ofinhibiting a polypeptide encoded by the polynucleotide sequencesaccording to the invention, wherein the inhibiting activity of thesemolecules is tested on at least said polypeptide, comprises the stepsof:

[0167] a) providing a polypeptide according to the invention;

[0168] b) contacting the active molecule with said polypeptide;

[0169] c) testing the capacity of the active molecules, at variousconcentrations, to inhibit the activity of the polypeptide; and

[0170] d) choosing the active molecule that provides an inhibitoryeffect of at least 80% on the activity of the said polypeptide.

[0171] A test for screening the inhibiting activity of a molecule, forexample, a new substrate analogue or a new antiparasitic agent, cancomprise the following steps:

[0172] A suitable test for testing an active molecule inhibiting thepolypeptide according to the invention is performed as follows:

[0173] The recombinant amino acid purified or native racemase is dilutedin sodium acetate buffer or Tris or phophate on Hepes buffer at 3micrograms per 500 microliters in the presence of 20 millimolar of betamercepto ethanol and 10 to 80 millimolar of L or D substrate andcontaining various concentrations of active molecule to be tested. Thisreaction is incubated for 30 minutes at 37° C. and stopped by heating at80° C. Variations in optical rotation are measured by a polarimeter.

[0174] Another embodiment of this invention provides a method forinhibiting the activity of a parasite in vivo. The method comprisesadministering to a host a parasite mitogen, which is capable ofexhibiting a protective effect, a curative effect, or preventingtransmission of a parasite in the host. The parasite mitogen isadministered to the host in an amount sufficient to prevent or at leastinhibit infection in vivo or to prevent or at least inhibit spread ofthe parasite in vivo. These effects are achieved by administering theparasite mitogen to the host in a sub-mitogenic amount, which ispreferably sufficient to induce a protective response against theparasite in the host.

[0175] The parasite mitogen employed in this invention is distinguishedfrom an “antigen”, which is a substance that induces an immune response,such as a complete antigen that both induces an immune response andreacts with the product of the response, or an incomplete antigen(hapten) that cannot induce an immune response by itself, but can reactwith the products of an immune response when complexed to a completeantigen (carrier). The parasite mitogens of the present invention arethus unlike antigens, which require processing and presentation, such as(1) uptake of the antigen by antigen presenting cells (APCs); (2)internalization of the antigen in intracellular vesicles; (3)intracellular processing, which may include the unfolding of a proteinand/or partial proteolysis, with generation of immunogenic peptides; (4)binding of peptides to class II MHC molecules to form a bimolecularcomplex recognized by T cells; and (5) transport to, and display of, thecomplex on the surface of APCs. In addition, the parasite mitogensemployed in this invention do not require activation of the APCs asmanifested by the expression of: (1) adhesion molecules that promote thephysical interaction between APCs and T cells; (2) membrane boundgrowth/differentiation molecules (co-stimulators) that promote T cellactivation; or (3) soluble cytokines, such as IL-1 and TNF, as isrequired in the process for presenting antigens.

[0176] The mitogen employed in this invention is also distinguished froma “superantigen”, which is a substance that can stimulate all of the Tcells in an individual that express a particular set or family of V_(β)Tcell receptor genes. Superantigens are typically bacterial and viralproducts, and can either be soluble or cell-bound. They do not requiredegradation to peptides. Superantigens are typically presented to the Tcell receptor (TCR) on MHC molecules; however, they do not requireprocessing by antigen presenting cells (APC), as do antigens, in orderto be presented.

[0177] Thus, used herein, the term “mitogen” refers to a polyclonalactivator that has the capacity to bind to and to trigger proliferationor differentiation of B lymphocytes, T lymphocytes, or mixtures thereof.Lymphocyte proliferation or transformation is the process whereby newDNA synthesis and cell division takes place in lymphocytes after astimulus of some type, resulting in a series of changes. The lymphocytesincrease in size, the cytoplasm becomes more extensive, the nucleoli arevisible in the nucleus, and the lymphocytes resemble blast cells. Theterm blast transformation is also sometimes applied to this process.Mitogens can induce proliferation in normals cells in culture.Activation of the lymphocytes thus can be characterized bytransformation of the lymphocytes into blast cells, synthesis of DNA,cell division, increased production of immunoglobulins, or increasedcytokine production. More particularly, the mitogens employed in thisinvention can stimulate whole classes of lymphocytes in this manner, andnot just clones of particular specificity. The mitogens employed in thisinvention function, therefore, in a manner similar to the effectsproduced by lipopolysaccharide (LPS) on B cells, or lectins,concanavalin A (ConA), and phytohemagglutinin (PHA) on T cells.

[0178] With these phenomena in mind, the expression “parasite mitogen”,as used herein, means at least one protein or polypeptide found in aparasite, wherein the protein or polypeptide is capable of provokingnon-specific polyclonal activation of B lymphocytes, T lymphocytes, ormixtures thereof, in an in vitro culture of the lymphocytes in themanner similar to that just described. The protein or polypeptidecomprising the parasite mitogen can be in glycosylated ornon-glycosylated form. The parasite mitogen can be in natural orrecombinant form.

[0179] The term “recombinant” as used herein means that a protein orpolypeptide employed in the invention is derived from recombinant (e.g.,microbial or mammalian) expression systems. “Microbial” refers torecombinant proteins or polypeptides made in bacterial or fungal (e.g.,yeast) expression systems. As a product, “recombinant microbial” definesa protein or polypeptide produced in a microbial expression system,which is essentially free of native endogenous substances. Proteins orpolypeptides expressed in most bacterial cultures, e.g. E. coli, will befree of glycan. Proteins or polypeptides expressed in yeast may have aglycosylation pattern different from that expressed in mammalian cells.

[0180] The parasite mitogen employed in this invention can be inisolated or purified form. The terms “isolated” or “purified”, as usedin the context of this specification to define the purity of protein orpolypeptide compositions, means that the protein or polypeptidecomposition is substantially free of other proteins of natural orendogenous origin and contains less than about 1% by mass of proteincontaminants residual of production processes. Such compositions,however, can contain other proteins added as stabilizers, excipients, orco-therapeutics. The parasite is isolated if it is detectable as asingle protein band in a polyacrylamide gel by silver staining.

[0181] Evaluation of lymphocyte proliferation can be quantitated in anassay of proliferative activity. For example, a radiolabelled precursorof DNA (usually tritiated thymidine) can be added to a culture mediumand the amount of radioactivity incorporated into the cells subsequentlydetected. A suitable assay involves the in vitro culture of a lymphocytepopulation in the presence or absence of a mitogen for various periodsof time. The changes induced in the stimulated groups are compared withchanges in unstimulated cell populations. Radiolabelled amino acids areconvenient as they provide a means of quantitating the changes in asimple, reproducible manner. Thus, as used herein, the expression “assayof proliferative activity” means the following assay:

Assay of Proliferative Activity

[0182] In vitro proliferation is accomplished using freshly recoveredsplenocytes from BALB/c mice seeded at a density of 5×10⁴ cells/well andincubated for 24, 48 and 72 h with increasing concentrations of totalparasite supernatants or recombinant TcPa45 protein or other mitogen(0.07-200 μg/ml) with 0.5 μg/ml of the HPLC fractions, or with theconventionally used mitogens concanavalin A (10 μg/ml) andlipopolysaccharide (5μg/ml) in 5% FCS in RPMI-1640 complete medium.T-cell depletion is accomplished by incubating freshly recovered spleencells for 30 min. at 37° C. in the presence of monoclonal antibodiesagainst Thy 1.2 and rabbit complement (Cedarlane, Le Perray en Yvelines,France). Analysis of proliferative activity of total splenocytes (5×10⁴cells/well) in the presence of 50 μg/ml enzymatically active rTcPA45 orother mitogen is also compared with the proliferation obtained using thesame amounts of rTcPA45 protein or other mitogen lacking racemaseactivity (by heating for 10 min. at 30° C. or by long term storage at 4°C.). Inhibition of proliferation is obtained by adding to the splenocytecultures 50 μg/ml rTcPA45 or other mitogen pre-incubated for 10 min. at37° C. with 1 mM inhibitor, either specific (pyrrole-2-carboxylic acid)or nonspecific (iodoacetamide or iodoacetate). Competitive assays ofproliferative activity by 50 μg/ml rTcPA45 or other mitogen are done byadding increasing concentrations of specific substrates i.e., prolineracemase substrates (L- or D-proline) for the mitogen rTcPA45 rangingfrom 3 mM to 50 mM. Controls include the incubation of splenocytes(5×10⁴ cells/well) with substrate alone, i.e., 50 mM L- or D-proline inRPMI medium alone for proline racemase. Cultures are collected after a16-hour pulse or 1 μCi/well ³H-thymidine uptake was determined in abeta-plate liquid scintillation counter (LKB-Wallac, Orsay, France). Alldata points are obtained in triplicate and the corresponding standarddeviation is calculated.

[0183] This assay of proliferative activity is used to determine whethera substance is a parasite mitogen. This assay is also used to determinea sub-mitogenic amount of the parasite mitogen. The results obtained ina typical assay of proliferative activity for two different mitogens, Aand B, are depicted in FIG. 13, which is not based on data actuallyobtained in this invention, but which is merely included forillustrative purposes to show the effects produced by mitogens afterlymphocyte activation.

[0184] Moreover, while the identification of a parasite or virus mitogenfor use in the invention is accomplished by use of the assay ofproliferative activity, it will be understood that the results obtainedwith this assay can be followed by other methods of measuring activationof lymphocytes, such as by measuring immunoglobulin production and/orcarrying out immunoglobulin specificity assays. The Elispot assaydescribed hereinafter can be used for this purpose.

[0185] As used herein, the term “sub-mitogenic amount” means an amountof the parasite mitogen, which is less than an amount of the parasitemitogen that produces an increase in lymphocyte proliferation in theassay of proliferative activity. Thus, the sub-mitogenic amount can beeasily determined by carrying out the assay of proliferative activity atseveral low dosages of the parasite mitogen and noting the dosage atwhich proliferative activity first increases. The sub-mitogenic amountis an amount below the dosage at which proliferative activity firstincreases.

[0186] The sub-mitogenic amount must also be sufficient to induceprotective immunity against the parasite in a host to which thesub-mitogenic amount of the parasite mitogen is administered. As usedherein, the term “protective immunity” refers to an adaptive (specific)immune response characterized by specificity and memory in the host towhich the sub-mitogenic amount of the parasite mitogen is administered.The adaptive immune response once stimulated by an invading parasitewill remember and respond more rapidly to infection so that no diseasewill occur or any disease that occurs following infection will be lesssevere as compared to a similar infection without prior immunizationaccording to the invention. Thus, the protective immunity imparted bythe method of the invention imparts protection from disease,particularly infectious disease, as evidenced by the absence of clinicalindications of disease, or as evidenced by absence of, or reduction in,determinants of pathogenicity, including the absence or reduction inpersistence of the infectious parasite or virus in vivo, and/or theabsence of pathogenesis and clinical disease, or diminished severitythereof, as compared to individuals not treated by the method of theinvention.

[0187] The determination of a sub-mitogenic amount can readily beunderstood by reference to FIG. 13, which depicts two dose-responsecurves for two different hypothetical mitogens A and B. A sub-mitogenicamount of mitogen A would be an amount below about dose “Y” in FIG. 13,while a sub-mitogenic amount of mitogen B would be an amount belowdosage “X” in FIG. 13.

[0188] The TcPA45 protein of the invention is referred to as a “parasitemitogen” in a functional sense in that it is capable of activating anon-specific polyclonal response in lymphocytes in the assay ofproliferative activity. TcPA45 itself might not be a mitogen, but mayact through racemization or by binding to host molecules, which would bethe primary mitogens. Regardless, the non-specific lymphocyte activationby TcPA45 in amounts exceeding a sub-mitogenic amount would insureevasion of the parasite at the very beginning of infection.

[0189] The evasiveness and diversity of parasites has made definitivetreatment difficult. Presented here are methods and agents forpreventing the spread of parasitic and viral infections in a host, suchas a human. Examples of microorganisms against which the methods andagents of the invention are effective are the following. TABLE 2 Immunedysfunctions observed after mitogen-induced polyclonal activationfollowing infectious processes Target lymph- Microorganism ocytes Acuteor progressive dysfunctions Actinomyces viscousus B ImmunosuppressionAfrican Swine Fever B Immunosuppression virus Ascaris B IgE secretin,allergy, cerebral granuloma Borrelia burgdorferi B Autoimmune arthritisCandida albicans B Granuloma formation, immunosuppression Chiamidiatrachomatis B Lymphocytosis, autoimmunity Entamoeba histolytica TImmunosuppression, disabling colitis, liver abscess Escherichia coil BToxic shock syndrome, meningitis, neurological and systemic symptomsLeishmania donovani, L B Immunosuppression, autoimmunity major Listeriamonocytogenes B and T Meningitis, immune complex formation andimmunosuppression Mycobacterium T Immunosuppression and tuberculosisautoimmune arthritis Plasmodium chabaudi, T Immunosuppression,autoimmunity P. yoellii P. falciparum B and T Immunosuppression,autoimmunity Salmonella paratyphi, S. B Lethal scepticemia, vasculartyphimurium myocardial injuries, immunosuppression, autoimmunitySchistosoma mansoni, S. B Immunosuppression, hematobium megasyndromes,granuloma Staphylococcus aureaus B Toxic shock syndrome, mastitis,immunosuppression Streptococcus B Toxic shock syndrome, intermedius, S.mutans immunosuppression S. pyogenes B and T Immunosuppression,autoimmunity Toxocara canis B Eosinophilia, lung damage, oculargranuloma, vasculitis Toxoplasma gondii B and T Encephalitis,myocarditis, immunosuppression Trypanosoma brucei B Immunosuppression,glomerulonephritis and brain lesions T. congolense B Immunosuppressin T.cruzi B and T Hypergammaglobulinemia, immunosuppression, autoimmunemyocarditis, megasyndromes

[0190] These and other substances employed as mitogens in this inventionare of parasitic or viral origin, are of natural or recombinant form,and are similarly capable of producing a polyclonal response inunselected lymphocytes in the assay of proliferative activity. Moreparticularly, while this invention has been described with reference toparasite mitogens, it is equally applicable to viral antigens. Indeed,by substituting “viral mitogen” for the expression “parasite mitogen”and “virus” for “parasite” in the foregoing description, the full scopeof the subject invention will be understood. In addition, the parasitemitogens employed in this invention will exemplified with reference tothe mitogen TcPA45 protein of T.cruzi. While the parasite mitogen TcPA45is also a racemase, it will be understood that the parasite mitogens andvirus mitogens can be employed in this invention may or may not possessracemase activity.

[0191] Following are additional examples of microorganisms against whichthe vaccination strategy of the invention is applicable: Infection withEpstein-Barr, influenza, herpes, and sendai viruses; non- or poorlypathogenic viruses, such as and African swine fever virus; and murineleukaemia virus. Both T-cell-dependent and independent polyclonal B-cellactivation have also been described for a variety of protozoan parasiteinfections, and these infections can also be prevented or abated by thevaccination strategy of this invention. These microorganisms includePlasmodium berghei, P. yoellii, P. chabaudi., P. falciparum and P.vivax.

[0192] In practicing the method of the invention, the parasite or viralmitogen is administered to a host using one of the modes ofadministration commonly employed for administering drugs to humans andother animals. Thus, for example, the parasite or viral mitogen can beadministered to the host by the oral route or parenterally, such as byintravenous or intramuscular injection. Other modes of administrationcan also be employed, such as intrasplenic, intradermal, and mucosalroutes. For purposes of injection, the mitogens described above can beprepared in the form of solutions, suspensions, or emulsions in vehiclesconventionally employed for this purpose.

[0193] It will be understood that the parasite and viral mitogens can beused in combination with other parasite or viral mitogens or otherprophylactic or therapeutic substances. For example, mixtures ofdifferent parasite mitogens or mixtures of different viral mitogens canbe employed in the method of the invention. Similarly, mixtures ofparasite and viral mitogens can be employed in the same composition. Theparasite and viral mitogens can also be combined with other vaccinatingagents for the corresponding disease, such microbial immunodominant,immunopathological and immunoprotective epitope-based vaccines orinactivated attenuated, or subunit vaccines. The parasite and viralmitogens can even be employed as adjuvants for other immunogenic orvaccinating agents.

[0194] The parasite or viral mitogen is employed in the method of theinvention in an amount sufficient to provide an adequate concentrationof the drug to prevent or at least inhibit infection of the host in vivoor to prevent or at least inhibit the spread of the parasite or virus invivo. The amount of the mitogen thus depends upon absorption,distribution, and clearance by the host. Of course, the effectiveness ofthe parasite or viral mitogen is dose related. The dosage of theparasite or viral mitogen should be sufficient to produce a minimaldetectable effect, but the dosage should be less than the dose thatactivates a non-specific polyclonal lymphocyte response as measured bythe assay of proliferative activity previously described.

[0195] The dosage of the parasite or viral mitogen administered to thehost can be varied over wide limits. The parasite or viral mitogen canbe administered in the minimum quantity, which is therapeuticallyeffective, and the dosage can be increased as desired up the maximumdosage tolerated by the patient. The parasite or viral mitogen can beadministered as a relatively high sub-mitogenic amount, followed bylower maintenance dose, or the parasite or viral mitogen can beadministered in uniform dosages.

[0196] The dosage and the frequency of administration will vary with theparasite or viral mitogen employed in the method of the invention. Inthe case of the TcPA45 parasite mitogen, the sub-mitogenic amountadministered to a human can vary from about 50 ng per Kg of body weightto about 1 μg per Kg of body weight, preferably about 100 ng per Kg ofbody weight to about 500 ng per Kg of body weight. Similar dosages canbe employed for the other parasite and viral mitogens employed in thisinvention but optimum amounts can be determined with a minimum ofexperimentation using conventional dose-response analytical techniquesor by scaling up from studies based on animal models of disease.

[0197] The term “about” as used herein in describing dosage ranges meansan amount that is equivalent to the numerically stated amount asindicated by the induction of protective immunity in the host to whichthe parasite or viral mitogen is administered, with the absence orreduction in the host of determinants of pathogenicity, including anabsence or reduction in persistence of the infectious parasite or virusin vivo, and/or the absence of pathogenesis and clinical disease, ordiminished severity thereof, as compared to individuals not treated bythe method of the invention.

[0198] The dose of the parasite or viral mitogen is specified inrelation to an adult of average size. Thus, it will be understood thatthe dosage can be adjusted by 20-25% for patients with a lighter orheavier build. Similarly, the dosage for a child can be adjusted usingwell known dosage calculation formulas.

[0199] The parasite or viral mitogen can be used in therapy in the formof pills, tablets, lozenges, troches, capsules, suppositories,injectable in ingestable solutions, and the like in the treatment ofcytopatic and pathological conditions in humans and susceptiblenon-human primates and other animals.

[0200] Appropriate pharmaceutically acceptable carriers, diluents, andadjuvants can be combined with the parasitic and viral mitogensdescribed herein in order to prepare the pharmaceutical compositions foruse in the treatment of pathological conditions in animals. Thepharmaceutical compositions of this invention contain the activemitogens together with a solid or liquid pharmaceutically acceptablenontoxic carrier. Such pharmaceutical carriers can be sterile liquids,such as water an oils, including those of petroleum, animal, vegetable,or synthetic origin. Examples of suitable liquids are peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Physiological solutions solutions can also be employed asliquid carriers, particularly for injectable solutions.

[0201] The ability of the vaccines of the invention to induce protectionin a host can be enhanced by emulsification with an adjuvant,incorporation in a liposome, coupling to a suitable carrier, or bycombinations of these techniques. For example, the vaccines of theinvention can be administered with a conventional adjuvant, such asaluminum phosphate and aluminum hydroxide gel. Similarly, the vaccinescan be bound to lipid membranes or incorporated in lipid membranes toform liposomes. The use of nonpyrogenic lipids free of nucleic acids andother extraneous matter can be employed for this purpose.

[0202] Suitable pharmaceutical excipients include starch, glucose,lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel,magnesium carbonate, magnesium stearate, sodium stearate, glycerolmonstearate, talc, sodium chloride, dried skim milk, glycerol, propyleneglycol, water, ethanol, and the like. These compositions can take theform of solutions, suspensions, tablets, pills, capsules, powders,sustained-release formulations and the like. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin. The pharmaceutical compositions contain an effective therapeuticamount of the parasite or viral mitogen together with a suitable amountof carrier so as to provide the form for proper administration to thehost.

[0203] The host or patient can be an animal susceptible to infection bythe parasite or virus, and is preferably a mammal. More preferably, themammal is selected from the group consisting of a human, a dog, a cat, abovine, a pig, and a horse. In an especially preferred embodiment, themammal is a human.

[0204] Another aspect of the invention includes administering nucleicacids encoding parasite and/or viral mitogens with or without carriermolecules to an individual. Those of skill in the art are cognizant ofthe concept, application, and effectiveness of nucleic acid vaccines(e.g., DNA vaccines) and nucleic acid vaccine technology as well asprotein and polypeptide based technologies. The nucleic acid basedtechnology allows the administration of nucleic acids encoding parasiteand/or viral mitogens, naked or encapsulated, directly to tissues andcells without the need for production of encoded proteins prior toadministration. The technology is based on the ability of these nucleicacids to be taken up by cells of the recipient organism and expressed toproduce a mitogen to which the recipient's immune system responds. Suchnucleic acid vaccine technology includes, but is not limited to,delivery of naked DNA and RNA and delivery of expression vectorsencoding the parasite or viral mitogen. Although the technology istermed “vaccine”, it is equally applicable to immunogenic compositionsthat do not result in a complete protective response. Suchpartial-protection-inducing compositions and methods are encompassedwithin the present invention.

[0205] Although it is within the present invention to deliver nucleicacids encoding the parasite or viral mitogens as naked nucleic acids,the present invention also encompasses delivery of nucleic acids as partof larger or more complex compositions. Included among these deliverysystems are viruses, virus-like particles, or bacteria containing thenucleic acids encoding the parasite or viral mitogen. Also, complexes ofthe invention's nucleic acids and carrier molecules with cellpermeabilizing compounds, such as liposomes, are included within thescope of the invention. Other compounds, such as molecular vectors (EP696,191, Samain et al.) and delivery systems for nucleic acid vaccinesare known to the skilled artisan and exemplified in, for example, WO 9306223 and WO 90 11092, U.S. Pat. No. 5,580,859, and U.S. Pat. No.5,589,466 (Vical patents), which are incorporated by reference herein,and can be made and used without undue or excessive experimentation.

[0206] Results indicate that intramuscular DNA vaccination protocolsusing pcDNA3 vector containing the TcPA45 gene, with or without thefragment encoding the signal peptide, are able to induce a decrease of85% in parasitemia levels after challenge with infective forms of theparasite. Moreover, even higher levels of parasitemia control resultedwhen sub-mitogenic doses of the active rTcPA45 protein were injectedintraperitoneally 2 weeks before infective challenge. These obervationssupport the use of this molecule as a drug and/or immunomodulatortarget.

[0207] This invention further contemplates:

[0208] 1. Any molecular modification of the gene or a fragment of thegene encoding for a racemase/mitogen that leads to the inhibition of theexpression of the protein by the parasite or virus (gene knock out), andfurther utilization of parasites or viruses lacking those activities invivo aiming at immunoprotective responses.

[0209] 2. Any molecular modification of the gene or a fragment of thegene encoding for a racemase/mitogen that leads to the hyperexpressionof the protein by the parasite or virus (gene transgenesis), and furtherutilization of the parasite or virus to produce high amounts of theprotein aiming at producing high amounts of the native protein.

[0210] 3. Any molecular modification of the gene or a fragment of thegene encoding for a racemase/mitogen that leads to an attenuation ofparasite or virus infectivity, or interaction with a host cell, andfurther injection of the parasites or viruses in vivo aiming atimmunoprotective responses.

[0211] 4. Any molecular modification (for instance directed mutagenesis)of the protein or of its active site that leads to the inhibition of itsenzymatic or its mitogenic activity and further injection of mutatedparasites or viruses in vivo aiming at immunoprotective responses.

[0212] 5. Use of any molecular or biochemical modification of theenzymatic activity of the racemase (inhibition of the active site)aiming at developing specific immunotherapy.

[0213] 6. Any molecule or compound that inhibits the enzymatic activityof the protein aiming at developing a drug against parasite or virusinfection or specific treatment of parasitic or viral disease.

[0214] An example of the application of this technology to the inventionfollows:

[0215] The catalytic site of TcPA45 protein responsible for racemaseactivity is identified by the boxed region in FIG. 2. The catalytic sitecomprises the amino acides SPCGT. Inhibition of racemase activity, andthe consequent loss in infectivity, can be accomplished by altering thiscatalytic site in the protein or altering the corresponding nucleotidesin the gene encoding the protein. A target for alteration would be thecysteine residue. For example, changing the cysteine residue to serinedoes not significantly alter the charge on the molecule, but diminishesracemase activity. The catalytic site can be altered in other ways, suchas by the addition, deletion, or substitution of another moiety for oneof the moieties in the protein or the nucleic acid encoding the proteinso that secondary and tertiary structures are not materially altered,binding sub-units are not affected, but racemase activity is diminishedor totally lost.

[0216] Plasmids containing the polynucleotides from T. Cruzi have beendeposited at the Collection Nationale de Cultures de Microorganismes(“C.N.C.M.”) Institut Pasteur, 28, rue du Docteur Roux, 75724 ParisCedex 15, France, as follows: Plasmid Accession No. Deposit DateDH5α-pTc45MIT (239 bp) I-2221 June 9, 1999 DH5α-pTc45MIT (1335 bp)I-2344 October 29, 1999.

[0217] This invention will now be described with reference to thefollowing Examples.

EXAMPLE 1 Mice and Parasites

[0218]Trypanosoma cruzi clone CL Brener was used throughout this work.Epimastigotes were maintained by weekly passage in liver infusiontryptose medium. In vitro metacyclogenesis was performed in a proteinfree defined medium at 27° C., as previously described¹¹. Male euthymicor athymic BALB/c mice 8 weeks of age were purchased from Charles RiverLaboratories (Saint Aubin les Elbeuf, France). Male C3H/Hel mice 8 weeksof age from our animal facilities were also used.

EXAMPLE 2 Protein Fractionation

[0219] 40 liters of culture supernatants from metacyclic forms,maintained for an additional 96 h at 37° C., were concentrated by vacuumdialysis and dialyzed against buffer A. HPLC was performed using a weakanion exchanger column POROS HQ-10 (Perspective Biosystems) at a flowrate of 1 ml/min according to the following program: a) 10 min withbuffer A; b) 30 min linear gradient from buffer A to B; c) 5 min lineargradient from buffer B to C; and d) 5 min with buffer C. One mlfractions were collected, frozen at −80° C., lyophilized andreconstituted in H₂O or in non-supplemented RPMI medium for in vitroproliferation assays. (Buffers used: A: 5 mM -NH₄-acetate, pH 8. B: 1MNH₄-acetate, pH 8. C: 1M NaCl/1 M NH₄-acetate, pH 8). Fractions 1 ml involume were collected, frozen at −80° C., lyophilized and reconstitutedin water or in non-supplemented RPMI medium for in vitro proliferationassays. SDS-PAGE analysis used standard techniques.

EXAMPLE 3 Generation of Peptides and Amino Acid Sequence Analysis

[0220] HPLC fractions 22 and 23 were pooled and fractionated by 8%SDS-PAGE. After amino black staining, the 45 kDa protein band was cutout, in-gel digested with trypsin, and submitted to reverse phase HPLCto separate peptides. Automated Edman degradation sequence analysis wasperformed in the Laboratoire de Microséquencage de Protéines of thePasteur Institut.

EXAMPLE 4 RNA Preparation, Reverse Transcription, PCR, and Cloning

[0221] RNA was extracted from trypomastigote forms obtained from Verocells, using TRIzol LS reagent (Gibco) following manufacturer'sinstructions. Two μg from this RNA were reverse transcribed in 20 μlwith Superscript II (Gibco) using anti-sense degenerate primer5′TTICCRMDATIACIACGTT3′ [SEQ ID NO: 12] designed from peptide 5.

[0222] PCR reaction was performed on 5 μl of cDNA using Taq polymerase(Perkin Elmer) or Pfu DNA polymerase (Stratagene). The following PCRconditions and primers were used: 1. TcPA45 gene fragment (239 bp) 30 sat 94° C., 45 s at 45° C., 30 s at 72° C. for 30 cycles followed by 10min at 72° C.; degenerate primers: corresponding to peptide 4 (forward)5′ATHGCITTYGGIGGIAAYTTT3′ [SEQ ID NO: 13] and to peptide 5 (reverse):5′TTICCRAADATIACIACGTT3′ [SEQ ID NO: 14]. (D for A, G or T; H stands forA, C or T; M for A or C; I for inosine; R for A or G; Y for C or T). 2.

[0223] The TcPA45 coding sequence (from codons 30 to 423) was amplifiedusing 45 s at 94° C., 45 s at 50° C., 3 min at 72° C. for 20 cycles,with primers 5′CTCTCCCATGGGGCAGGAAAAGCTTCTG3′ [SEQ ID NO: 15] and5′CTGAGCTCGACCAGATCTATCTGC3′ [SEQ ID NO: 16]. PCR products were purifiedwith Qiagen PCR extraction kit and cloned into pCR II-TOPO vector usingthe TOPO-TA cloning kit (Invitrogen) following manufacturer'sinstructions.

EXAMPLE 5 Automated Sequencing

[0224] Lambda phage and plasmid DNA were prepared using standardtechniques, and direct sequencing was performed using Big Dye Terminatorkit (Perkin Elmer) following manufacturer's instructions. Extensionproducts were run for 7 h in an ABI 373B automated sequencer. Primersinternal to the sequence have also been used for sequencing.

EXAMPLE 6 Genomic Library Screening

[0225] A genomic library of T. cruzi CL-Brener constructed in phagelambda Fix II (from Dr. E. Rondinelli, UFRJ, Brazil) was screened usinga ³²P labelled 239 bp PCR product as a probe. Hybridization wasperformed using standard conditions. Filters were scanned using aPhosporImager scanning unit (Molecular Dynamics). Positive phages wereidentified and phage DNA was prepared using standard procedures.

EXAMPLE 7 Expression Constructs and Recombinant Protein Expression

[0226] The PCR product encoding the TcPA45 gene fragment starting atcodon 30 was cloned in frame with a C-terminal 6× histidine tag into thepET28b(+) expression vector (Novagen). The soluble recombinant proteinwas produced in E. coli, and the soluble fraction was purified using aNi²⁺ column (Novagen) following manufacturer's instructions.

EXAMPLE 8 Racemase Activity

[0227] Demonstration of the racemase enzymatic activity of rTcPA 45 useda polarimeter. Buffer Na-acetate (pH 6), reaction Vol. 500 pl. OptimumpH was found to be 6 and the temperature 37° C.

[0228] 3 μg of rTcPA45 was diluted in 500 μl of buffer (0.2M NaOAc, 20mM β-mercaptoethanol, pH 6.0) containing different concentrations of thesubstrate (10-80 mM or L- or D-proline). (See FIG. 4c.) The reaction wasincubated for 30-60 minutes at 37° C. in a water bath, followed by heatinactivation of the enzyme for 10 minutes at 80° C. Each sample isdiluted to 1.5 ml with water and the mixture submitted to measurement ofoptical rotation using a polarimeter, at 365 nm. A sample without enzymeis used as a control. For optimal pH determination, buffer systems withNaOAc, phosphate, and Tris were used with pH ranging from 4.0-8.2.Combined results are shown in FIG. 4e. Curve of temperature wasperformed between 27° C. to 47° C.

EXAMPLE 9 Mitogenic Activity

[0229] Analysis of mitogenic proliferative activity of spleen cells invitro with rTcPA45 (His/tag) protein.

[0230] The figure [FIG. 4] is representative of 3 experiments usingdifferent mouse strains (C57BL/6, BLAB/c and C3H:HeJ, the latter aLPS-nonresponsive strain). Similar results were obtained with the 3strains, including C3H/HeJ, showing that our preparation is notcontaminated by bacterial LPS. The proliferation is dose-dependent andpresents a bi-modal pattern as already observed with total culturesupernatants used to purify the Tc45 protein. Best cellconcentration=5×10⁴ cells/well.

[0231] 5×10⁵ naive spleen cells/well (96 well plate) were stimulated invitro with different doses of rTcPA45 (ranging in FIG. 4b from 0.8 to200 μg/ml final) for 24, 48 and 72 h, at 37° C., 5% CO₂. Cultures werepulsed with ³H-Thymidine (1 μCi/well) for 16-18 h before harvesting.³H-thymidine incorporation was obtained after counting using abeta-plate. Results present arithmetic means of c.p.m. (counts/minute)from 6 wells/dose of rTcPA45 or wells containing medium alone (+/−SD ofthe means). (FIG. 4b)

EXAMPLE 10 Mitogenicity of rTcPA45 in Vivo Assessed by ELISPOT.

[0232] BALB/c mice were injected or not with 50 μg of rTcPA45 (i.p.),and spleen cells assayed day 7 after injection. Results represent totalnumbers of spleen cells, total number of B cells producing IgM, IgG2a,or IgG2b isotypes, and total numbers of isotype-producing B cellsspecific of the protein. TABLE 1 ELISPOT assay 7 days after i.p.injection of rTc45MIT Total Number of Ig-producing B cells IgM-producingIgG2a- IgG2b- cells producing cells producing cells Non-injected 163000± 25456 3650 ± 636  4300 ± 142 PTc45MIT 371666 ± 94495 9866 ± 3000 14633± 3287 (50 μg/mouse)

[0233] Total Number of Ig-producing B cells ANTI-rTc45MIT IgM-B IgG2a-IgG2b- cells anti 45 cells anti 45 cells anti 45 Non-injected none nonenone PTc45MIT 84 none none (50 μg/mouse)

[0234] It is observed 1) a 2 fold increase in total spleen cell numbersafter 7 days of rTcPA45 injection, 2) 3-6 fold increase in total numbersof Ig-secreting cells, and 3) less than 0.5% of IgM secreting cells aredirected to the injected protein, characterizing a mitogenic stimulationof B cells.

[0235] Mitogenicity of the rTcPA45 in vivo (assay)

[0236] Mice were injected or not with 50 μg of iTcPA45 (i.p.). 7 dayslater, spleens were removed and cell suspensions were prepared andcounted. Numbers of Ig-secreting cells (total or specific to rTcPA45)were determined by Elispot assay, as follows:

[0237] ELISPOT Assay

[0238] Flat bottomed 96 well ELISA plates, were coated with either goatanti-mouse Ig or with rTcPA45 and incubated at 4° C. overnight. Plateswere blocked with PBS-gelatin, washed with PBS-Tween and with RPMImedium. 100 μl of different spleen cell concentrations per well (in RPMIcontaining 2% FCS) were incubated for 8 hours at 37° C., 5% CO₂. A rowin the plate containing serial dilutions of purified immunoglobulin(IgM, IgG2a, or IgG2b) was used as standard. After lysis of the cellswith H₂O-Tween, plates were then washed with PBS-Tween and incubatedwith respective biotin-labeled antibodies directed to (IgM, IgG2b, orIgG2a isotypes), overnight at 4° C. After washing, plates were incubatedfor 45-60 minutes with avidin-alkaline phosphatase and further incubatedwith substrate (2-amino-2-methyl-propanol buffer containing BCIP) 2-4hours, 37° C. (until the spots are “dark” blue). Spots correspond toimmunoglobulin-producing B-cell (of a particular isotype) directed tothe coated antigen (here: goat anti-mouse immunoglobulins or rTcPA45).Spots are then counted and numbers corrected to total number of spleencells according to the dilution. (See Table I and FIG. 7.)

EXAMPLE 11 DNA Vaccination

[0239] 8 week old BALB/c mice (5 mice/group) were injected (i.m.) once,or 3 times (interval of 3 weeks) with the different constructions (100μl plasmid/femoral quadriceps), as follows:

[0240] a) controls: saline and rTc24

[0241] b) Vectors (pcDNA3 and VR1020, which is described by R. Amasamyet al., Biochemica Biophysica Acta 1998, Vol, 1453, pp. 1-13) of DNAvaccination containing different constructs: Long, containing thecomplete sequence of rTcPA45 gene; Short, containing the sequence ofrTcPA gene without the signal peptide, *VR1020 vector contains anadditional signal peptide (tissue Plasminogen Activating factor, TPA).

[0242] c) empty vectors

[0243] Mice were challenged 4 weeks after the last injection with 10⁴infective forms of the parasite/mouse, and the parasitemia was scoredduring 35 days. Serum samples were collected before challenge andassayed by Western blot against the recombinant protein.

[0244] It is worth noting that BALB/c mice were treated by almost 2months (9 weeks) to follow the vaccination protocol and were challengedat 21 week old. It is well known that over 10 week old mice areresistant to the experimental infection with Trypanosoma cruzi and nomortality is observed.

[0245] The results using this vaccination protocol revealed that 3injections of pcDNA3 containing either the Short or Long constructs, orjust 1 injection of the same vector with the Short construct is able toreduce by more than 50% the parasitemia levels. Titres of totalimmunoglobulins anti-rTcPA45, 4 weeks after the last plasmidinjection:controls (saline and empty vectors): 1:100; both constructs inpcDNA3 (Short and Long): 1:2000, and respectively 1/1000 and 1:2000 forVR1020 containing the Long and Short sequences. (See FIG. 8.)

EXAMPLE 12 Southern, Western and Northern Blots

[0246] Mice were immunized intrasplenically with 10 ng protein and wereboosted every 3 weeks with 1 μg of the same preparation for 2 months toobtain polyclonal serum containing rTcPA45-specific antibodies. Total,soluble and insoluble sonic extracts, or culture supernatants from thedifferent parasite forms were purified and separated by 8-10% SDS-PAGE,and proteins were electrophoretically transferred to nitrocellulosemembranes. Membranes were saturated with Tris-buffered saline and milk,incubated with polyclonal serum against rTcPA45 and developed withperoxidase-labeled secondary antibody using an ECL kit (Arnersham,Orsay, France). T. cruzi genomic DNA (10 μg) was digested withrestriction enzymes (BarntII, BglII, SalI, TaqI and PstI), separated by0.8% agarose gel electrophoresis and transferred to Hyband N+ followedby hybridization of the membrane with a ³³P-dATP-labeled probe coveringthe TcPA45-coding sequence. Total RNA was prepared from epimastigote,metacyclic and trypomastigote forms of the parasite by conventionalmethods. For northern blot analysis, 20 μg epimastigote RNA wastransferred to Hybond N+ membranes, then hybridized with single-strandedDNA complementary to the TcPA45 gene transcript, labeled withα-³²P-dCTP.

[0247] Transcript analysis through reverse transcription and PCR. TotalRNA (1 μg) from epimastigote, metacyclic and trypomastigote forms of theparasite were used to synthesize specific first-strand cDNA by usingoligonucleotide R300-45 (5′-TCCGTATCCATGTCGATGC-3′) [SEQ ID NO:24],located about 240 nucleotides downstream from the first ATG start codon,followed by PCR amplification using R300-45 and an oligonucleotidecorresponding to part of the T cruzi spliced leader sequence(5′-TATTATTGATACAGTTTCTG-3′) [SEQ ID NO:25]. An internal TcPA45 fragmentof about 170 bp was then amplified using R300-45 and the oligonucleotideHI-45 (5′-CTCTCCCATGGGGCAGGAAAAGCTTCTG-3′) [SEQ ID NO:26] to demonstratethe presence of Tc45 transcript in each of the life stages analyzed.

EXAMPLE 13 Immunofluorescence

[0248] Cellular localization of TcPA45 protein in epimastigote,metacyclic and bloodstream forms of the parasite was demonstrated byindirect immunofluorescence using polyclonal mouse serum against rTcPA45(described above) followed by 4 μg/ml Alexa 488™ goat antibody againstmouse IgG (H+I), F(ab′)₂ fragment conjugate (Molecular Probes-Interchim,Montlucon, France), compared with control staining using Alexa 488™F(ab′)₂ fragment conjugate alone or after incubation of the parasiteswith chronic serum obtained from mice infected for 8 months.

EXAMPLE 14 Racemization Assays

[0249] The percent of racemization of different concentrations ofL-proline, D-proline, L-hydroxy-proline and D-hydroxy-proline substrateswas calculated by incubating a 500-μl mixture of 3 μg TcPA45 and 10-80mM substrate in 0.2 M sodium acetate and 25 mM β-mercaptoethanol, pH 6,for 30 min at 37° C. The reaction was stopped by incubation for 10 minat 80° C. Water (1 ml) was then added, and the optical rotation wasmeasured in polarimeter 241 MC (Perkin Elmer, Montignyle Bretonneux,France) at a wavelength of 365 nm, in a cell with a path length of 10cm. The percent inhibition of racemization of 80 mM L-proline wasdetermined in the presence of different concentrations of severalspecific and nonspecific inhibitors ranging in concentration from 6 mMto 100 mM. The percent racemization of 80 mM L-proline as a function ofpH was determined using 0.2 M sodium acetate, postassium phosphate andTris-HCl buffers containing 25 mM β-mercaptoethanol; reactions wereincubated for 30 min at 37° C. as described above. All reagents andinhibitors were purchased from Sigma.

[0250] Accession numbers. The GenBank accession number of T. cruziTcPA45 is AF195522. The EMBL accession number of C. sticklandii isE10199.

EXAMPLE 15

[0251] rTcPA45 is a B cell mitogen from a pathogenic trypanosome and anovel eukaryotic proline racemase. Both mitogenic and racemaseactivities seem to be linked and dependent on the integrity, or theavailability of the enzyme active site. Specific inhibition of theactive site (using specific or non-specific proline racemaseinhibitors), or the active site occupancy by the substrate (competitionassays using L- or D-proline), respectively, abolish or decrease B-cellmitogenic properties of rTcPA45.

[0252] As with classical B cell mitogens, rTcPA45 mitogenicity isdose-dependent (FIG. 1). As for classical B cell mitogens, mousespecific immune responses directed to mitogenic rTcPA45 is indeedpossible (Western blot FIG. 12) following a protocol of immunizationwhich consisted of with 1 injection (i.p.) of a sub-mitogenic (10ng/mouse) dose of the protein followed by an additional boost with amitogenic dose of rTcPA45 (50 μg/mouse) one week later. Protocol

[0253] 5 groups of 6 week old Balb/c mice (3 mice per group) wereimmunized as follows:

[0254] T=non immunized.

[0255] Vide=immunized with 50 μg of empty PcDNA3 vector, i.m. per 3 week(twice).

[0256] Court=immunized with 50 μg of PcDNA3−short (TcPA45 short), i.m.per week (twice).

[0257] I. S.=immunized with 10 ng of rTcPA45 i.s. (first week) and 50 μgrTcPA45 i.p. (second week).

[0258] I.P.=immunized with 10 ng of rTcPA45 i.p. (first week) and 50 μgrTcPA45 i.p. (second week).

[0259] Sera from individual mice were collected and analyzed by Westernblot:

[0260] 80 μg of rTcPA45 were loaded onto 0.8% SDS-page gels andtransferred to Hybond membranes.

[0261] Sera were individually tested at {fraction (1/400)} dilution andreactivity against rTcPA45 revealed with anti-IgG mice immunoglobulinsusing chemuluminescence.

[0262]FIG. 12 (J-14) represents individual serum reactivity beforeimmunization before challenge (J0), and after 10 days (J10) or 21 days(J21) of challenge. Challenge consisted of i.p 10000 parasites.

[0263] Additionally, rTcPA45 molecule can be considered as a target forvaccination strategies, since the previous Example using DNA-vaccinationprotocols (intramuscular injections of DNA-vectors containing the Tc45gene) was able to reduce by 85% the parasitemia levels after aninfectious challenge with the parasite. (See FIG. 14a.)

[0264] Moreover, experiments showed that higher levels of parasitemiacontrol (90-95%) was obtained when the submitogenic protocol ofimmunization described above was followed by an infectious challenge(10⁴ parasites/mouse). These additional results do support the claimthat mitogenic moieties are potential targets for vaccination approachesagainst Trypanosoma cruzi infection. (See FIG. 14(b).)

[0265] In summary, we have investigated the mechanisms and consequencesof polyclonal lymphocyte activation using the experimental model ofChagas disease, caused by the protozoan parasite T. cruzi. As in otherinfectious processes, this disease involves extensive B- and T-cellactivation, hypergammaglobulinemia, and the establishment of chronicautoimmunity affecting the heart and the digestive tract. Trypanosomacruzi infection induces a lymphocyte blast transformation of a magnitudethat is similar to or higher than that induced by the classic polyclonalactivator LPS.

[0266] This invention involves the use of a parasite or viral mitogen asa vaccinating agent without lymphocyte polyclonal activation thatinhibits a protective host specific immune response to the parasite orvirus. This invention not only prevents infection by the parasite orvirus, but also avoids the negative consequences of such infection(immunosuppression, persistent infection, and susceptibility toimmunopathology and autoimmune phenomenon). This invention thusaddresses the correlation of polyclonal lymphocyte activation of theimmune system after infections with poor specific responses and severeimmunosuppression to autologous or unrelated challenges through aneffective vaccination strategy.

REFERENCES

[0267] The following publications are cited herein. The entiredisclosure of each publication is relied upon and incorporated byreference herein.

[0268] 1. Reina-San-Martin, B., Cosson, A. & Minoprio, P. Lymphocytepolyclonal activation: a pitfall for vaccine design against infectiousagents. Parasitol. Today 16(2); 62-67 (2000).

[0269] 2. Minoprio, P., Itohara, S., Heusser, C., Tonegawa, S. &Coutinho, A. Immunobiology of murine T. cruzi infection: thepredominance of parasite-nonspecific responses and the activation ofTcRI T cells Immunol. Rev. 112,183-207 (1989).

[0270] 3. Eisen, H., Petry, K. & Voorhis, W. V. The origin of theautoimmune pathology associated with Trypanosoma cruzi infection(Academic Press, Inc., New York, 1990).

[0271] 4. Ribeiro-dos-Santos, R., et al. Anti-CD4 abrogates rejectionand reestablishes long-term tolerance to syngeneic newborn heartsgrafted in mice chronically infected with Trypanosoma cruzi J. Exp. Med.175, 29-39 (1992).

[0272] 5. Tarleton, R., Zhang, L. & Downs, M. Autoimmune rejection ofneonatal heart transplants in experimental Chagas' disease is aparasite-specific response to infected host tissue. P.N.A.S. 94,3932-3937 (1997).

[0273] 6. Arala-Chaves, M., d'Imperio-Lima, M. R., Coutinho, A.,Pena-Rossi, C. & Minoprior, P. V-region-related and -unrelatedimmunosuppression accompanying infections Mem. Inst. Osw. Cruz 87, 35-41(1992).

[0274] 7. Minoprio, P., Eisen, H., Joskowicz, M., Pereira, P. &Coutinho, A. Suppression of polyclonal antibody production inTrypanosoma cruzi infected mice by treatment with anti-L3T4 antibodies.J. Immunol. 139, 545-550 (1987).

[0275] 8. Minoprio, P., Coutinho, A., Spinella, S. &Hontebeyrie-Joskowicz, M. Xid immunodeficiency imparts increasedparasite clearance and resistance to pathology in experimental Chagas'disease Internat. Immunol. 3, 427-433 (1991).

[0276] 9. Santos-Lima, E. C. & Minoprio, P. Chagas' disease isattenuated in mice lacking γδ T cells Infec. Immun. 64, 215-221 (1996).

[0277] 10. Cordeiro, da Silva, A., Guevara Espinoza, A., Taibi, A.,Ouaissi, A. & Minoprio, P. A 24 kDa Trypanosoma cruzi antigen is a Bcell activator Immunology 94, 189-196 (1998).

[0278] 11. Contreras, V. T., Salles, J. M., Thomas, N., Morel, C. M. &Goldenberg, S. In vitro differentiation of Trypanosoma cruzi underchemically defined conditions. Mol. Biochem. Parasitol. 16, 315-327(1985).

[0279] 12. Dragon, E. A., Sias, S. R., Kato, E. A. & Gabe, J. D. Thegenome of Trypanosoma cruzi contains a constitutively expressed,tandemly arranged multicopy gene homologous to a major heat shockprotein. Mol. Cell. Biol. 7, 1271-1275 (1987).

[0280] 13. Moro, A., Ruiz-Cabello, F., Fernandez-Cano, A., Stock, R. P.& Gonzalez, A. Secretion by Trypanosoma cruzi of a peptidyl-prolylcis-trans isomerase involved in cell infection. EMBO Journal 14(11),2483-2490 (1995).

[0281] 14. Cardinale, G. J. & Abeles, R. H. Purification and mechanismof action of proline racemase. Biochemistry 7(11), 3970-3978 (1968).

[0282] 15. Rudnick, G. & Abeles, R. Reaction mechanism and structure ofthe active site of proline racemase. Biochem. 14(20), 4515-4522 (1975).

[0283] 16. Lamzin, V. S., Zbigniew, D. & Wilson, K. S. How nature dealswith stereoisomers. Curr. Op. Struct. Biol. 5, 830-836 (1995).

[0284] 17. Barret, F. M. Changes in the concentration of free aminoacids in the haemolymph of Rhodnius prolixus during the fifth instar.Comp. Biochem. Physiol. 48(B), 241-250 (1973).

[0285] 18. Sylvester, D. & Krassner, S. M. Proine metabolism inTrypanosoma cruzi epimastigotes. Comp. Biochem. Physiol. 55(B), 443-447(1976).

[0286] 19. de Isola, E. L., Lammel, E. M., Katzin, V. J. & GonzalezCappa, S. M. Influence of organ extracts of Triatoma infestans ondifferentiation of Trypanosoma cruzi. J. Parasitol. 67(1), 53-58 (1981).

[0287] 20. Rosenberg, R. D., et al. Heparan sulfate proteoglycans of thecardiovascular system: specific structures emerge but how is synthesisregulated ? J. Clin. Inv. 100(11S), 67S-75S (1997).

[0288] 21. Herrera, E. M., Ming, M., Ortega-Barria, E. & Pereira, M. E.Mediation of Trypanosoma cruzi invasion by heparan sulfate receptors onhost cells and penetrin counter receptors on the trypanosomes. Mol.Biochem. Parasitol. 75(1), 73-83 (1994).

[0289] 22. Ortega-Barria, E. & Pereira, M. E. A novel T. cruzi heparinbinding protein promotes fibroblast adhesion and penetration ofengineered bacteria and trypanosomes into mammalian cells. Cell 67,411-421 (1991).

[0290] 23. Shakibaei, M. & Frevert, U. Dual interaction of the malariacircunsporozoite protein with the low density lipoprotein receptorrelated (LRP) and heparan sulfate proteoglycans. J. Exp. Med. 184,1699-1711 (1996).

[0291] 24. Thompson, R. J., Bouwer, H. G., Portnoi, D. A. & Frankel, F.R. Pathogenicity and immunogenicity of a Listeria monocytogenes strainthat requires D-alanine for growth. Inf. Immun. 66(8), 3552-3561 (1998).

[0292] 25. Mozes, E., Sela, M. & Taussig, M. J. Tolerance to thymusindependent antigens. Characteristics of induction of tolerance tothymus independent synthetic polypeptides. Immunol. 27, 641-646 (1974).

[0293] 26. Sela, M. & Zisman, E. Different rols of D-amino acids inimmune phenomena. FASEB J. 11, 449-456 (1997).

[0294] 27. Coutinho, A. & Moller, G. B cell mitogenic properties ofthymus-independent antigens Nature 245,12-14 (1973).

[0295] 28. Coutinho, A., Gronowicz, E., Bullock, W. W. & Moller, G.Mechanism of thymus-independent immunocyte triggering. Mitogenicactivation of B cells results in specific immune responses. J. Exp. Med.139(1), 74-92 (1974).

[0296] 29. Janeway, C. A. & Humphrey, J. H. Synthetic antigens composedexclusively of L- or D-amino acids. II. Effect of optical configurationon the metabolism and fate of synthetic polypeptide antigens in mice.Folia Biol. 16(3), 156-172 (1970).

[0297] 30. d'Imperio-Lima, M. R., Eisen, H., Minoprio, P., Joskowicz, M.& Coutinho, A. Persistance of polyclonal B cell activation withundetectable parasitemia in late stages of experimental Chagas' disease.J. Immunol. 137, 353-356 (1986).

[0298] 31. Tavares, D., Ferreira, P., Vilanova, M., Videira, A. &Arala-Chaves, M. Immunoprotection against systemic candidiasis in mice.Int. Immunol. 7(5), 785-96 (1995).

[0299] 32. in D-amino acids in sequences of secreted peptides ofmulticellular organisms. (eds. Jolles, P.) (Birkhauser Verlag, Basel,1998).

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1 26 1 418 PRT Trypanosoma cruzi 1 Met Arg Lys Ser Val Cys Pro Lys GlnLys Phe Phe Phe Ser Ala Phe 1 5 10 15 Pro Phe Phe Phe Phe Phe Cys ValPhe Pro Leu Ile Ser Arg Thr Gly 20 25 30 Gln Glu Lys Leu Leu Phe Asp GlnLys Tyr Lys Ile Ile Lys Gly Glu 35 40 45 Lys Lys Glu Lys Lys Lys Asn GlnArg Ala Asn Arg Arg Glu His Gln 50 55 60 Gln Lys Arg Glu Ile Met Arg PheLys Lys Ser Phe Thr Cys Ile Asp 65 70 75 80 Met His Thr Glu Gly Glu AlaAla Arg Ile Val Thr Ser Gly Leu Pro 85 90 95 His Ile Pro Gly Ser Asn MetAla Glu Lys Lys Ala Tyr Leu Gln Glu 100 105 110 Asn Met Asp Tyr Leu ArgArg Gly Ile Met Leu Glu Pro Arg Gly His 115 120 125 Asp Asp Met Phe GlyAla Phe Leu Phe Asp Pro Ile Glu Glu Gly Ala 130 135 140 Asp Leu Gly MetVal Phe Met Asp Thr Gly Gly Tyr Leu Asn Met Cys 145 150 155 160 Gly HisAsn Ser Ile Ala Ala Val Thr Ala Ala Val Glu Thr Gly Ile 165 170 175 ValSer Val Pro Ala Lys Ala Thr Asn Val Pro Val Val Leu Asp Thr 180 185 190Pro Ala Gly Leu Val Arg Gly Thr Ala His Leu Gln Ser Gly Thr Glu 195 200205 Ser Glu Val Ser Asn Ala Ser Ile Ile Asn Val Pro Ser Phe Leu Tyr 210215 220 Gln Gln Asp Val Val Val Val Leu Pro Lys Pro Tyr Gly Glu Val Arg225 230 235 240 Val Asp Ile Ala Phe Gly Gly Asn Phe Phe Ala Ile Val ProAla Glu 245 250 255 Gln Leu Gly Ile Asp Ile Ser Val Gln Asn Leu Ser ArgLeu Gln Glu 260 265 270 Ala Gly Glu Leu Leu Arg Thr Glu Ile Asn Arg SerVal Lys Val Gln 275 280 285 His Pro Gln Leu Pro His Ile Asn Thr Val AspCys Val Glu Ile Tyr 290 295 300 Gly Pro Pro Thr Asn Pro Glu Ala Asn TyrLys Asn Val Val Ile Phe 305 310 315 320 Gly Asn Arg Gln Ala Asp Arg GlyThr Ser Ala Lys Met Ala Thr Leu 325 330 335 Tyr Ala Lys Gly Gln Leu ArgIle Gly Glu Thr Phe Val Tyr Glu Ser 340 345 350 Ile Leu Gly Ser Leu PheGln Gly Arg Val Leu Gly Glu Glu Arg Ile 355 360 365 Pro Gly Val Lys ValPro Val Thr Lys Asp Ala Glu Glu Gly Met Leu 370 375 380 Val Val Thr AlaGlu Ile Thr Gly Lys Ala Phe Ile Met Gly Phe Asn 385 390 395 400 Thr MetLeu Phe Asp Pro Thr Asp Pro Phe Lys Asn Gly Phe Thr Leu 405 410 415 LysGln 2 389 PRT Trypanosoma cruzi 2 Arg Thr Gly Gln Glu Lys Leu Leu PheAsp Gln Lys Tyr Lys Ile Ile 1 5 10 15 Lys Gly Glu Lys Lys Glu Lys LysLys Asn Gln Arg Ala Asn Arg Arg 20 25 30 Glu His Gln Gln Lys Arg Glu IleMet Arg Phe Lys Lys Ser Phe Thr 35 40 45 Cys Ile Asp Met His Thr Glu GlyGlu Ala Ala Arg Ile Val Thr Ser 50 55 60 Gly Leu Pro His Ile Pro Gly SerAsn Met Ala Glu Lys Lys Ala Tyr 65 70 75 80 Leu Gln Glu Asn Met Asp TyrLeu Arg Arg Gly Ile Met Leu Glu Pro 85 90 95 Arg Gly His Asp Asp Met PheGly Ala Phe Leu Phe Asp Pro Ile Glu 100 105 110 Glu Gly Ala Asp Leu GlyMet Val Phe Met Asp Thr Gly Gly Tyr Leu 115 120 125 Asn Met Cys Gly HisAsn Ser Ile Ala Ala Val Thr Ala Ala Val Glu 130 135 140 Thr Gly Ile ValSer Val Pro Ala Lys Ala Thr Asn Val Pro Val Val 145 150 155 160 Leu AspThr Pro Ala Gly Leu Val Arg Gly Thr Ala His Leu Gln Ser 165 170 175 GlyThr Glu Ser Glu Val Ser Asn Ala Ser Ile Ile Asn Val Pro Ser 180 185 190Phe Leu Tyr Gln Gln Asp Val Val Val Val Leu Pro Lys Pro Tyr Gly 195 200205 Glu Val Arg Val Asp Ile Ala Phe Gly Gly Asn Phe Phe Ala Ile Val 210215 220 Pro Ala Glu Gln Leu Gly Ile Asp Ile Ser Val Gln Asn Leu Ser Arg225 230 235 240 Leu Gln Glu Ala Gly Glu Leu Leu Arg Thr Glu Ile Asn ArgSer Val 245 250 255 Lys Val Gln His Pro Gln Leu Pro His Ile Asn Thr ValAsp Cys Val 260 265 270 Glu Ile Tyr Gly Pro Pro Thr Asn Pro Glu Ala AsnTyr Lys Asn Val 275 280 285 Val Ile Phe Gly Asn Arg Gln Ala Asp Arg GlyThr Ser Ala Lys Met 290 295 300 Ala Thr Leu Tyr Ala Lys Gly Gln Leu ArgIle Gly Glu Thr Phe Val 305 310 315 320 Tyr Glu Ser Ile Leu Gly Ser LeuPhe Gln Gly Arg Val Leu Gly Glu 325 330 335 Glu Arg Ile Pro Gly Val LysVal Pro Val Thr Lys Asp Ala Glu Glu 340 345 350 Gly Met Leu Val Val ThrAla Glu Ile Thr Gly Lys Ala Phe Ile Met 355 360 365 Gly Phe Asn Thr MetLeu Phe Asp Pro Thr Asp Pro Phe Lys Asn Gly 370 375 380 Phe Thr Leu LysGln 385 3 29 PRT Trypanosoma cruzi 3 Met Arg Lys Ser Val Cys Pro Lys GlnLys Phe Phe Phe Ser Ala Phe 1 5 10 15 Pro Phe Phe Phe Phe Phe Cys ValPhe Pro Leu Ile Ser 20 25 4 354 PRT Trypanosoma cruzi 4 Met Arg Phe LysLys Ser Phe Thr Cys Ile Asp Met His Thr Glu Gly 1 5 10 15 Glu Ala AlaArg Ile Val Thr Ser Gly Leu Pro His Ile Pro Gly Ser 20 25 30 Asn Met AlaGlu Lys Lys Ala Tyr Leu Gln Glu Asn Met Asp Tyr Leu 35 40 45 Arg Arg GlyIle Met Leu Glu Pro Arg Gly His Asp Asp Met Phe Gly 50 55 60 Ala Phe LeuPhe Asp Pro Ile Glu Glu Gly Ala Asp Leu Gly Met Val 65 70 75 80 Phe MetAsp Thr Gly Gly Tyr Leu Asn Met Cys Gly His Asn Ser Ile 85 90 95 Ala AlaVal Thr Ala Ala Val Glu Thr Gly Ile Val Ser Val Pro Ala 100 105 110 LysAla Thr Asn Val Pro Val Val Leu Asp Thr Pro Ala Gly Leu Val 115 120 125Arg Gly Thr Ala His Leu Gln Ser Gly Thr Glu Ser Glu Val Ser Asn 130 135140 Ala Ser Ile Ile Asn Val Pro Ser Phe Leu Tyr Gln Gln Asp Val Val 145150 155 160 Val Val Leu Pro Lys Pro Tyr Gly Glu Val Arg Val Asp Ile AlaPhe 165 170 175 Gly Gly Asn Phe Phe Ala Ile Val Pro Ala Glu Gln Leu GlyIle Asp 180 185 190 Ile Ser Val Gln Asn Leu Ser Arg Leu Gln Glu Ala GlyGlu Leu Leu 195 200 205 Arg Thr Glu Ile Asn Arg Ser Val Lys Val Gln HisPro Gln Leu Pro 210 215 220 His Ile Asn Thr Val Asp Cys Val Glu Ile TyrGly Pro Pro Thr Asn 225 230 235 240 Pro Glu Ala Asn Tyr Lys Asn Val ValIle Phe Gly Asn Arg Gln Ala 245 250 255 Asp Arg Ser Pro Cys Gly Thr GlyThr Ser Ala Lys Met Ala Thr Leu 260 265 270 Tyr Ala Lys Gly Gln Leu ArgIle Gly Glu Thr Phe Val Tyr Glu Ser 275 280 285 Ile Leu Gly Ser Leu PheGln Gly Arg Val Leu Gly Glu Glu Arg Ile 290 295 300 Pro Gly Val Lys ValPro Val Thr Lys Asp Ala Glu Glu Gly Met Leu 305 310 315 320 Val Val ThrAla Glu Ile Thr Gly Lys Ala Phe Ile Met Gly Phe Asn 325 330 335 Thr MetLeu Phe Asp Pro Thr Asp Pro Phe Lys Asn Gly Phe Thr Leu 340 345 350 LysGln 5 330 PRT Clostridium sticklandii 5 Met Lys Phe Ser Lys Gly Ile HisAla Ile Asp Ser His Thr Met Gly 1 5 10 15 Glu Pro Thr Arg Ile Val ValGly Gly Ile Pro Gln Ile Asn Gly Glu 20 25 30 Thr Met Ala Asp Lys Lys LysTyr Leu Glu Asp Asn Leu Asp Tyr Val 35 40 45 Arg Thr Ala Leu Met His GluPro Arg Gly His Asn Asp Met Phe Gly 50 55 60 Ser Ile Ile Thr Ser Ser AsnAsn Lys Glu Ala Asp Phe Gly Ile Ile 65 70 75 80 Phe Met Asp Gly Gly GlyTyr Leu Asn Met Cys Gly His Gly Ser Ile 85 90 95 Gly Ala Ala Thr Val AlaVal Glu Thr Gly Met Val Glu Met Val Glu 100 105 110 Pro Val Thr Asn IleAsn Met Glu Ala Pro Ala Gly Leu Ile Lys Ala 115 120 125 Lys Val Met ValGlu Asn Glu Lys Val Lys Glu Val Ser Ile Thr Asn 130 135 140 Val Pro SerPhe Leu Tyr Met Glu Asp Ala Lys Leu Glu Val Pro Ser 145 150 155 160 LeuAsn Lys Thr Ile Thr Phe Asp Ile Ser Phe Gly Gly Ser Phe Phe 165 170 175Ala Ile Ile His Ala Lys Glu Leu Gly Val Lys Val Glu Thr Ser Gln 180 185190 Val Asp Val Leu Lys Lys Leu Gly Ile Glu Ile Arg Asp Leu Ile Asn 195200 205 Glu Lys Ile Lys Val Gln His Pro Glu Leu Glu His Ile Lys Thr Val210 215 220 Asp Leu Val Glu Ile Tyr Asp Glu Pro Ser Asn Pro Glu Ala ThrTyr 225 230 235 240 Lys Asn Val Val Ile Phe Gly Gln Gly Gln Val Asp ArgGly Thr Ser 245 250 255 Ala Lys Leu Ala Thr Leu Tyr Lys Lys Gly His LeuLys Ile Asp Glu 260 265 270 Lys Glu Val Tyr Glu Ser Ile Thr Gly Thr MetPhe Lys Gly Arg Val 275 280 285 Leu Glu Glu Thr Lys Val Gly Glu Phe AspAla Ile Ile Pro Glu Ile 290 295 300 Thr Gly Gly Ala Tyr Ile Thr Gly GluAsn His Glu Val Ile Asp Pro 305 310 315 320 Glu Asp Pro Leu Lys Tyr GlyPhe Thr Val 325 330 6 314 PRT Pseudomonas aeruginosa 6 Met Gln Arg IleArg Ile Ile Asp Ser His Thr Gly Gly Glu Pro Thr 1 5 10 15 Arg Leu ValIle Gly Gly Phe Pro Asp Leu Gly Gln Gly Asp Met Ala 20 25 30 Glu Arg ArgArg Leu Leu Gly Glu Arg His Asp Ala Trp Arg Ala Ala 35 40 45 Cys Ile LeuGlu Pro Arg Gly Ser Asp Val Leu Val Gly Ala Leu Leu 50 55 60 Cys Ala ProVal Asp Pro Glu Ala Cys Ala Gly Val Ile Phe Phe Asn 65 70 75 80 Asn SerGly Tyr Leu Gly Met Cys Gly His Gly Thr Ile Gly Leu Val 85 90 95 Ala SerLeu Ala His Leu Gly Arg Ile Gly Pro Gly Val His Arg Ile 100 105 110 GluThr Pro Val Gly Glu Val Glu Ala Thr Leu His Glu Asp Gly Ser 115 120 125Val Ser Val Arg Asn Val Pro Ala Tyr Arg Tyr Arg Arg Gln Val Ser 130 135140 Val Glu Val Pro Gly Ile Gly Arg Val Ser Gly Asp Ile Ala Trp Gly 145150 155 160 Gly Asn Trp Phe Phe Leu Val Ala Gly His Gly Gln Arg Leu AlaGly 165 170 175 Asp Asn Leu Asp Ala Leu Thr Ala Tyr Thr Val Ala Val GlnGln Ala 180 185 190 Leu Asp Asp Gln Asp Ile Arg Gly Glu Asp Gly Gly AlaIle Asp His 195 200 205 Ile Glu Leu Phe Ala Asp Asp Pro His Ala Asp SerArg Asn Phe Val 210 215 220 Leu Cys Pro Gly Lys Ala Tyr Asp Arg Ser ProCys Gly Thr Gly Thr 225 230 235 240 Ser Ala Lys Leu Ala Cys Leu Ala AlaAsp Gly Lys Leu Leu Pro Gly 245 250 255 Gln Pro Trp Arg Gln Ala Ser ValIle Gly Ser Gln Phe Glu Gly Arg 260 265 270 Tyr Glu Trp Leu Asp Gly GlnPro Gly Gly Pro Ile Val Pro Thr Ile 275 280 285 Arg Gly Arg Ala His ValSer Ala Glu Ala Thr Leu Leu Leu Ala Asp 290 295 300 Asp Asp Pro Phe AlaTrp Gly Ile Arg Arg 305 310 7 1665 DNA Trypanosoma cruzi 7 cctttttctttttaaaaaca aaaaaaattc cggggggaat atggaacagg gtatatgcgt 60 aaaagtgtctgtcccaaaca aaaatttttt ttttccgcct tcccattttt tttttttttt 120 tgtgtgtttcccttgatctc tcgaacaggg caggaaaagc ttctgtttga ccaaaaatat 180 aaaattattaagggcgagaa aaaagaaaag aaaaaaaatc aacgagcaaa caggagagaa 240 caccaacaaaaaagggaaat tatgcgattt aagaaatcat tcacatgcat cgacatgcat 300 acggaaggtgaagcagcacg gattgtgacg agtggtttgc cacacattcc aggttcgaat 360 atggcggagaagaaagcata cctgcaggaa aacatggatt atttgaggcg tggcataatg 420 ctggaaccacgtggtcatga tgatatgttt ggagcctttt tatttgaccc tattgaagaa 480 ggcgctgacttgggcatggt attcatggat accggtggct atttaaatat gtgtggacat 540 aactcaattgcagcggttac ggcggcagtt gaaacgggaa ttgtgagcgt gccggcgaag 600 gcaacaaatgttccggttgt cctggacaca cctgcggggt tggtgcgcgg tacggcacac 660 cttcagagtggtactgagag tgaggtgtca aatgcgagta ttatcaatgt accctcattt 720 ttgtatcagcaggatgtggt ggttgtgttg ccaaagccct atggtgaagt acgggttgat 780 attgcatttggaggcaattt tttcgccatt gttcccgcgg agcagttggg aattgatatc 840 tccgttcaaaacctctccag gctgcaggag gcaggagaac ttctgcgtac tgaaatcaat 900 cgcagtgtgaaggttcagca ccctcagctg ccccatatta acactgtgga ctgtgttgag 960 atatacggtccgccaacgaa cccggaggca aactacaaga acgttgtgat atttggcaat 1020 cgccaggcggatcgctctcc atgtgggaca ggcaccagcg ccaagatggc aacactttat 1080 gccaaaggccagcttcgcat cggagagact tttgtgtacg agagcatact cggctcactc 1140 ttccagggcagggtacttgg ggaggagcga ataccggggg tgaaggtgcc ggtgaccaaa 1200 gatgccgaggaagggatgct cgttgtaacg gcagaaatta ctggaaaggc ttttatcatg 1260 ggtttcaacaccatgctgtt tgacccaacg gatccgttta agaacggatt cacattaaag 1320 cagtagatctggtagagcac agaaactatt ggggaacacg tgcgaacagg tgctgctacg 1380 tgaagggtattgaatgaatc gttttttttt atttttattt tttattttta ttagtgcatt 1440 attattaaattttttttttg ttttggggtt tcaacggtac cgcgttggga gcagggaagc 1500 gatagcggccggacaatttt ttgcttttat tttcattttc atcttcctac ccaaccccct 1560 tggttccaccggtcgcggcg gggtcttgtg ggtggaggag tcctaaatcc cgcacctcgg 1620 aggaataaacatatttcaat ttcatatctt ggaatcaaaa ggcat 1665 8 1575 DNA Trypanosoma cruzi8 ttttccgcct tcccattttt tttttttttt tgtgtgtttc ccttgatctc tcgaacaggg 60caggaaaagc ttctgtttga ccaaaaatat aaaattatta agggcgagaa aaaagaaaag 120aaaaaaaatc aacgagcaaa caggagagaa caccaacaaa aaagggaaat tatgcgattt 180aagaaatcat tcacatgcat cgacatgcat acggaaggtg aagcagcacg gattgtgacg 240agtggtttgc cacacattcc aggttcgaat atggcggaga agaaagcata cctgcaggaa 300aacatggatt atttgaggcg tggcataatg ctggaaccac gtggtcatga tgatatgttt 360ggagcctttt tatttgaccc tattgaagaa ggcgctgact tgggcatggt attcatggat 420accggtggct atttaaatat gtgtggacat aactcaattg cagcggttac ggcggcagtt 480gaaacgggaa ttgtgagcgt gccggcgaag gcaacaaatg ttccggttgt cctggacaca 540cctgcggggt tggtgcgcgg tacggcacac cttcagagtg gtactgagag tgaggtgtca 600aatgcgagta ttatcaatgt accctcattt ttgtatcagc aggatgtggt ggttgtgttg 660ccaaagccct atggtgaagt acgggttgat attgcatttg gaggcaattt tttcgccatt 720gttcccgcgg agcagttggg aattgatatc tccgttcaaa acctctccag gctgcaggag 780gcaggagaac ttctgcgtac tgaaatcaat cgcagtgtga aggttcagca ccctcagctg 840ccccatatta acactgtgga ctgtgttgag atatacggtc cgccaacgaa cccggaggca 900aactacaaga acgttgtgat atttggcaat cgccaggcgg atcgctctcc atgtgggaca 960ggcaccagcg ccaagatggc aacactttat gccaaaggcc agcttcgcat cggagagact 1020tttgtgtacg agagcatact cggctcactc ttccagggca gggtacttgg ggaggagcga 1080ataccggggg tgaaggtgcc ggtgaccaaa gatgccgagg aagggatgct cgttgtaacg 1140gcagaaatta ctggaaaggc ttttatcatg ggtttcaaca ccatgctgtt tgacccaacg 1200gatccgttta agaacggatt cacattaaag cagtagatct ggtagagcac agaaactatt 1260ggggaacacg tgcgaacagg tgctgctacg tgaagggtat tgaatgaatc gttttttttt 1320atttttattt tttattttta ttagtgcatt attattaaat tttttttttg ttttggggtt 1380tcaacggtac cgcgttggga gcagggaagc gatagcggcc ggacaatttt ttgcttttat 1440tttcattttc atcttcctac ccaaccccct tggttccacc ggtcgcggcg gggtcttgtg 1500ggtggaggag tcctaaatcc cgcacctcgg aggaataaac atatttcaat ttcatatctt 1560ggaatcaaaa ggcat 1575 9 1524 DNA Trypanosoma cruzi 9 cgaacagggcaggaaaagct tctgtttgac caaaaatata aaattattaa gggcgagaaa 60 aaagaaaagaaaaaaaatca acgagcaaac aggagagaac accaacaaaa aagggaaatt 120 atgcgatttaagaaatcatt cacatgcatc gacatgcata cggaaggtga agcagcacgg 180 attgtgacgagtggtttgcc acacattcca ggttcgaata tggcggagaa gaaagcatac 240 ctgcaggaaaacatggatta tttgaggcgt ggcataatgc tggaaccacg tggtcatgat 300 gatatgtttggagccttttt atttgaccct attgaagaag gcgctgactt gggcatggta 360 ttcatggataccggtggcta tttaaatatg tgtggacata actcaattgc agcggttacg 420 gcggcagttgaaacgggaat tgtgagcgtg ccggcgaagg caacaaatgt tccggttgtc 480 ctggacacacctgcggggtt ggtgcgcggt acggcacacc ttcagagtgg tactgagagt 540 gaggtgtcaaatgcgagtat tatcaatgta ccctcatttt tgtatcagca ggatgtggtg 600 gttgtgttgccaaagcccta tggtgaagta cgggttgata ttgcatttgg aggcaatttt 660 ttcgccattgttcccgcgga gcagttggga attgatatct ccgttcaaaa cctctccagg 720 ctgcaggaggcaggagaact tctgcgtact gaaatcaatc gcagtgtgaa ggttcagcac 780 cctcagctgccccatattaa cactgtggac tgtgttgaga tatacggtcc gccaacgaac 840 ccggaggcaaactacaagaa cgttgtgata tttggcaatc gccaggcgga tcgctctcca 900 tgtgggacaggcaccagcgc caagatggca acactttatg ccaaaggcca gcttcgcatc 960 ggagagacttttgtgtacga gagcatactc ggctcactct tccagggcag ggtacttggg 1020 gaggagcgaataccgggggt gaaggtgccg gtgaccaaag atgccgagga agggatgctc 1080 gttgtaacggcagaaattac tggaaaggct tttatcatgg gtttcaacac catgctgttt 1140 gacccaacggatccgtttaa gaacggattc acattaaagc agtagatctg gtagagcaca 1200 gaaactattggggaacacgt gcgaacaggt gctgctacgt gaagggtatt gaatgaatcg 1260 tttttttttatttttatttt ttatttttat tagtgcatta ttattaaatt ttttttttgt 1320 tttggggtttcaacggtacc gcgttgggag cagggaagcg atagcggccg gacaattttt 1380 tgcttttattttcattttca tcttcctacc caaccccctt ggttccaccg gtcgcggcgg 1440 ggtcttgtgggtggaggagt cctaaatccc gcacctcgga ggaataaaca tatttcaatt 1500 tcatatcttggaatcaaaag gcat 1524 10 87 DNA Trypanosoma cruzi 10 atgcgtaaaagtgtctgtcc caaacaaaaa tttttttttt ccgccttccc attttttttt 60 tttttttgtgtgtttccctt gatctct 87 11 1395 DNA Trypanosoma cruzi 11 aagaaatcattcacatgcat cgacatgcat acggaaggtg aagcagcacg gattgtgacg 60 agtggtttgccacacattcc aggttcgaat atggcggaga agaaagcata cctgcaggaa 120 aacatggattatttgaggcg tggcataatg ctggaaccac gtggtcatga tgatatgttt 180 ggagcctttttatttgaccc tattgaagaa ggcgctgact tgggcatggt attcatggat 240 accggtggctatttaaatat gtgtggacat aactcaattg cagcggttac ggcggcagtt 300 gaaacgggaattgtgagcgt gccggcgaag gcaacaaatg ttccggttgt cctggacaca 360 cctgcggggttggtgcgcgg tacggcacac cttcagagtg gtactgagag tgaggtgtca 420 aatgcgagtattatcaatgt accctcattt ttgtatcagc aggatgtggt ggttgtgttg 480 ccaaagccctatggtgaagt acgggttgat attgcatttg gaggcaattt tttcgccatt 540 gttcccgcggagcagttggg aattgatatc tccgttcaaa acctctccag gctgcaggag 600 gcaggagaacttctgcgtac tgaaatcaat cgcagtgtga aggttcagca ccctcagctg 660 ccccatattaacactgtgga ctgtgttgag atatacggtc cgccaacgaa cccggaggca 720 aactacaagaacgttgtgat atttggcaat cgccaggcgg atcgctctcc atgtgggaca 780 ggcaccagcgccaagatggc aacactttat gccaaaggcc agcttcgcat cggagagact 840 tttgtgtacgagagcatact cggctcactc ttccagggca gggtacttgg ggaggagcga 900 ataccgggggtgaaggtgcc ggtgaccaaa gatgccgagg aagggatgct cgttgtaacg 960 gcagaaattactggaaaggc ttttatcatg ggtttcaaca ccatgctgtt tgacccaacg 1020 gatccgtttaagaacggatt cacattaaag cagtagatct ggtagagcac agaaactatt 1080 ggggaacacgtgcgaacagg tgctgctacg tgaagggtat tgaatgaatc gttttttttt 1140 atttttattttttattttta ttagtgcatt attattaaat tttttttttg ttttggggtt 1200 tcaacggtaccgcgttggga gcagggaagc gatagcggcc ggacaatttt ttgcttttat 1260 tttcattttcatcttcctac ccaaccccct tggttccacc ggtcgcggcg gggtcttgtg 1320 ggtggaggagtcctaaatcc cgcacctcgg aggaataaac atatttcaat ttcatatctt 1380 ggaatcaaaaggcat 1395 12 20 DNA Artificial Sequence Description of ArtificialSequence Primer 12 ttnccraada tnacnacgtt 20 13 21 DNA ArtificialSequence Description of Artificial Sequence Primer 13 athgcnttyggnggnaaytt t 21 14 20 DNA Artificial Sequence Description of ArtificialSequence Primer 14 ttnccraada tnacnacgtt 20 15 28 DNA ArtificialSequence Description of Artificial Sequence Primer 15 ctctcccatggggcaggaaa agcttctg 28 16 24 DNA Artificial Sequence Description ofArtificial Sequence Primer 16 ctgagctcga ccagatctat ctgc 24 17 1665 DNATrypanosoma cruzi 17 cctttttctt tttaaaaaca aaaaaaattc cggggggaatatggaacagg gtatatgcgt 60 aaaagtgtct gtcccaaaca aaaatttttt ttttccgccttcccattttt tttttttttt 120 tgtgtgtttc ccttgatctc tcgaacaggg caggaaaagcttctgtttga ccaaaaatat 180 aaaattatta agggcgagaa aaaagaaaag aaaaaaaatcaacgagcaaa caggagagaa 240 caccaacaaa aaagggaaat tatgcgattt aagaaatcattcacatgcat cgacatgcat 300 acggaaggtg aagcagcacg gattgtgacg agtggtttgccacacattcc aggttcgaat 360 atggcggaga agaaagcata cctgcaggaa aacatggattatttgaggcg tggcataatg 420 ctggaaccac gtggtcatga tgatatgttt ggagcctttttatttgaccc tattgaagaa 480 ggcgctgact tgggcatggt attcatggat accggtggctatttaaatat gtgtggacat 540 aactcaattg cagcggttac ggcggcagtt gaaacgggaattgtgagcgt gccggcgaag 600 gcaacaaatg ttccggttgt cctggacaca cctgcggggttggtgcgcgg tacggcacac 660 cttcagagtg gtactgagag tgaggtgtca aatgcgagtattatcaatgt accctcattt 720 ttgtatcagc aggatgtggt ggttgtgttg ccaaagccctatggtgaagt acgggttgat 780 attgcatttg gaggcaattt tttcgccatt gttcccgcggagcagttggg aattgatatc 840 tccgttcaaa acctctccag gctgcaggag gcaggagaacttctgcgtac tgaaatcaat 900 cgcagtgtga aggttcagca ccctcagctg ccccatattaacactgtgga ctgtgttgag 960 atatacggtc cgccaacgaa cccggaggca aactacaagaacgttgtgat atttggcaat 1020 cgccaggcgg atcgctctcc atgtgggaca ggcaccagcgccaagatggc aacactttat 1080 gccaaaggcc agcttcgcat cggagagact tttgtgtacgagagcatact cggctcactc 1140 ttccagggca gggtacttgg ggaggagcga ataccgggggtgaaggtgcc ggtgaccaaa 1200 gatgccgagg aagggatgct cgttgtaacg gcagaaattactggaaaggc ttttatcatg 1260 ggtttcaaca ccatgctgtt tgacccaacg gatccgtttaagaacggatt cacattaaag 1320 cagtagatct ggtagagcac agaaactatt ggggaacacgtgcgaacagg tgctgctacg 1380 tgaagggtat tgaatgaatc gttttttttt atttttattttttattttta ttagtgcatt 1440 attattaaat tttttttttg ttttggggtt tcaacggtaccgcgttggga gcagggaagc 1500 gatagcggcc ggacaatttt ttgcttttat tttcattttcatcttcctac ccaaccccct 1560 tggttccacc ggtcgcggcg gggtcttgtg ggtggaggagtcctaaatcc cgcacctcgg 1620 aggaataaac atatttcaat ttcatatctt ggaatcaaaaggcat 1665 18 4 PRT Trypanosoma cruzi 18 Trp Ile Ile Lys 1 19 11 PRTTrypanosoma cruzi 19 Ile Val Thr Gly Ser Leu Pro Asp Ile Ser Gly 1 5 1020 16 PRT Trypanosoma cruzi 20 Ala Thr Asn Val Pro Val Val Leu Asp ThrPro Ala Gly Leu Val Arg 1 5 10 15 21 9 PRT Trypanosoma cruzi 21 Val AspIle Ala Phe Gly Gly Asn Phe 1 5 22 8 PRT Trypanosoma cruzi 22 Asn ValVal Ile Phe Gly Asn Arg 1 5 23 7 PRT Trypanosoma cruzi 23 Met Ala ThrLeu Tyr Ala Lys 1 5 24 19 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide 24 tccgtatcca tgtcgatgc 19 25 20 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide25 tattattgat acagtttctg 20 26 28 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide 26 ctctcccatg gggcaggaaa agcttctg 28

What is claimed is:
 1. A purified nucleic acid molecule selected fromthe group consisting of SEQ ID NOS: 7, 8, 9, 10, and
 11. 2. A purifiednucleic acid molecule encoding a peptide selected from the groupconsisting of of SEQ ID NOS: 1, 2, 3, and
 4. 3. A purified nucleic acidmolecule that hybridizes to either strand of a denatured,double-stranded DNA comprising the nucleic acid molecule of any one ofclaims 1 or 2 under conditions of moderate stringency.
 4. The purifiednucleic acid molecule as claimed in claim 3, wherein said isolatednucleic acid molecule is derived by in vitro mutagenesis from SEQ IDNOS: 7, 8, 9, 10, and
 11. 5. A purified nucleic acid molecule degeneratefrom SEQ ID NOS: 7, 8, 9, 10, and 11 as a result of this genetic code.6. A purified nucleic acid molecule, which encodes Tc45 polypeptide, anallelic variant of Tc45 polypeptide, or a homolog of Tc45 polypeptide.7. A recombinant vector that directs the expression of a nucleic acidmolecule selected from the group consisting of the purified nucleic acidmolecules of claims 1, 2, 4, 5, and
 6. 8. A recombinant vector thatdirects the expression of a nucleic acid molecule of claim
 3. 9. Arecombinant vector that directs the expression of a nucleic acidmolecule of claim
 4. 10. A purified polypeptide encoded by a nucleicacid molecule selected from the group consisting of the purified nucleicacid molecules of claims 1, 2, 3, 4, 5, and
 6. 11. A purifiedpolypeptide according to claim 10 having a molecular weight ofapproximately 45 kDa as determined by SDS-PAGE.
 12. A purifiedpolypeptide according to claim 11 in post translationally modified formor not.
 13. A purified polypeptide encoded by a nucleic acid molecule ofclaim
 3. 14. A purified polypeptide according to claim 13 in posttranslationally modified form or not.
 15. A purified polypeptide encodedby a nucleic acid molecule of claim
 4. 16. A purified polypeptideaccording to claim 15 in post translationally modified form or not. 17.A purified eukaryotic amino acid racemase having a 38 kd to 45 kda moreor less 10%.
 18. Purified antibodies that bind to a polypeptide of claim10.
 19. Purified antibodies according to claim 17, wherein theantibodies are monoclonal antibodies.
 20. Purified antibodies that bindto a polypeptide of claim
 13. 21. Purified antibodies according to claim19, wherein the antibodies are monoclonal antibodies.
 22. Purifiedantibodies that bind to a polypeptide of claim
 15. 23. Purifiedantibodies according to claim 21, wherein the antibodies are monoclonalantibodies.
 24. A host cell transfected or transduced with the vector ofclaim
 7. 25. A method for the production of Tc45 polypeptide comprisingculturing a host cell of claim 24 under conditions promoting expression,and recovering the polypeptide from the host cell or the culture medium.26. The method of claim 25, wherein the host cell is selected from thegroup consisting of bacterial cells, parasite cells and eukaryoticcells.
 27. A host cell transfected or transduced with the vector ofclaims 6 to
 9. 28. A method for the production of Tc45 polypeptidecomprising culturing a host cell of claim 24 under conditions promotingexpression, and recovering the polypeptide from the host cell or theculture medium.
 29. The method of claim 28, wherein the host cell isselected from the group consisting of bacterial cells, parasite cellsand eukaryotic cells.
 30. A host cell transfected or transduced with thevector of claim
 9. 31. A method for the production of Tc45 polypeptidecomprising culturing a host cell of claim 30 under conditions promotingexpression, and recovering the polypeptide from the host cell or theculture medium.
 32. The method of claim 31, wherein the host cell isselected from the group consisting of bacterial cells, or parasiticcells or eukaryotic cells.
 33. The plasmid deposited at CNCM under theAccession Number I-2221 or I-2344.
 34. An immunological complexcomprising a Tc45 polypeptide and an antibody that specificallyrecognizes said polypeptide.
 35. A method of detecting a parasite in abiological sample that harbors a polynucleotide sequence according toclaim 1, said method comprising the steps of: (a) contacting parasiteDNA of the biological sample with a primer or a probe, which hybridizeswith the polynucleotide sequence of claim 1; (b) amplifying thenucleotide sequence using said primer or said probe; and (c) detecting ahybridized complex formed between said primer or probe and the DNA. 36.A method of detecting a parasite in a biological sample that harbors apolypeptide according to claims 10 to 16 or fragments or peptidesthereof, which can be recognized by antibodies raised against a P38 toP45 kDa racemase, said method comprising the steps of: (a) contactingthe parasite extract or the biological sample with antibodies accordingto any one of claims 18 to 23; and (b) detecting the resultingimmunocomplex.
 37. A kit for detecting a parasite that harbors apolynucleotide sequence according to claim 1, said kit comprising: (a) apolynucleotide probe, which hybridizes with the polynucleotide sequenceof claim 1; and (b) reagents to perform a nucleic acid hybridizationreaction.
 38. A kit according to claim 37 comprising: (a) purifiedantibodies according to any one of claims 18 to 23, which react with thepolypeptide as claimed in any one of claims 10 to 16; (b) standardreagents as the parasite racemase in a purified form; and (c) detectionreagents.
 39. An in vitro method of screening for active moleculescapable of inhibiting a polypeptide encoded by a polynucleotide sequenceaccording to any one of claims 1 to 6, said method comprising the stepsof: (a) contacting the active molecules with said polypeptide; (b)testing the capacity of the active molecules, at various concentrations,to inhibit the activity of the polypeptide; and (c) choosing the activemolecule that provides an inhibitory effect of at least 80% on theactivity of the said polypeptide.
 40. A purified polynucleotide encodingan eukaryotic protein with an amino acid racemase activity.
 41. Apurified polynucleotide encoding an eukaryotic protein with a prolineracemase activity.
 42. A polynucleotide encoding an eukaryotic protein,which is recognized by antibodies raised against an eukaryotic proteinhaving an amino acid racemase activity such as a proline racemase.
 43. Apolynucleotide according to claim 37 or 38 having at least 80% ofidentity with the sequence of an eukaryotic gene encoding a protein witha racemase activity.
 44. A method of detecting an eukaryotic proteinencoded by a polynucleotide as claimed in claim 40 or 41 comprising thesteps of: (a) contacting the sample with antibodies raised against anamino acid racemase such as proline racemase; and (b) detecting theresulting immunocomplex.
 45. A kit for detecting a parasite thatexpresses a polypeptide encoded by a polynucleotide sequence accordingto claim 1, said kit comprising: (a) antibodies raised against an aminoacid racemase, such as proline racemase; and (b) reagents for thedetection of an immunocomplex.
 46. A method of detection andquantification of presence or absence of a polynucleotide sequenceaccording to any one of claims 1 to 6, or a sequence hybridizing undermoderate strinigency with a polynucleotides, according to any one ofclaims 40, 41, or 42, or a fragment thereof.
 47. A fragment of apolynucleotide containing at least 50 nucleotides of the sequence of theproline racemase gene of T. cruzi or hybridizing under stringentconditions with a polynucleotide according to any one of claims 40, 41,42, or
 43. 48. A purified eukaryotic protein with an amino acid racemaseactivity such as a proline racemase.
 49. A purified P38 to P45 kDaprotein according to claim
 48. 50. Purified P38 to P45 kDa according toany one of claims 48 or 49, which is a parasite protein.
 51. PurifiedP38 to P45 kDa protein according to claim 50, wherein the parasite is T.cruzi.
 52. Purified antibodies against an eukaryotic racemase accordingto any one of claims 48 or
 49. 53. A process of preparation of apurified eukaryotic protein with a racemase activity comprising thefollowing steps: selecting a gene encoding a protein having a racemaseactivity; transforming a host with a recombinant vector containing thegene; culturing the host and producing the protein encoded by the gene;separating the purified eukaryotic protein with the racemase activityfrom the culture; or separating the purified eukaryotic proteinrecognized by antibodies raised against the protein claimed in any oneof claims 48, 49, 50, or
 51. 54. A process for detecting a T cruziinfection by contacting purified P45 and fragments or peptides thereof,which are recognized by antibodies raised against a polypeptide claimedin any one of claims 10 to 16, with serum of a patient suspected to beinfected.
 55. An immunizing composition containing at least a purifiedprotein according to any one of claims 48, 49, 50, or 51, or a fragmentthereof, capable of inducing an immune response in vivo.
 56. Animmunizing composition containing at least a purified protein accordingto any one of claims 48, 49, 50 and 51, or a fragment thereof, capableto induce the inhibition of a mitogenic polyclonal immunoresponse invivo.
 57. An immunizing composition against a parasite infectioncontaining at least the purified protein according to any one of claims48, 49, 50, or 51, or a fragment thereof.
 58. A vaccine compositionagainst a T. cruzi infection containing the purified 38 to P45 kdaprotein or a fragment thereof according to claim
 51. 59. A process forscreening a molecule capable of inhibiting the amino acid racemaseactivity of an eukaryotic protein comprising the steps of: contactingthe purified eukaryotic racemase protein with standard doses of amolecule to be tested; measuring inhibition of racemase activity; andselecting the molecule.
 60. A method of inhibiting an eukaryotic proteinwith an amino acid racemase activity, which comprises treating a patientby administering an effective amount of a molecule that inhibits saideukaryotic protein.
 61. Method according to claim 60, wherein theparasite is T. cruzi.
 62. A method for producing an eukaryoticrecombinant amino acid racemase comprising the following steps: (a)culturing a bacterial or a eukaryotic host harboring an over expressionsystem including an insert containing a polynucleotide sequence encodingan eukaryotic amino acid racemase; (b) separating the recombinanteukaryotic amino acid racemase from the host proteins; and (c) purifyingthe eukaryotic amino acid racemase.
 63. A method according to claim 62,wherein the amino acid racemase is a proline racemase.
 64. A methodaccording to claim 62, wherein the recombinant bacterial host containsan insert derived from the insert contained in the strain deposited atCNCM under Accession number I-2344.
 65. A method for the production ofD-amino acid using a purified eukaryotic amino acid racemase comprisingthe steps of: (a) incubating L-amino acid with the recombinanteukaryotic amino acid racemase; (b) separating the D-amino acid producedin step a; and (c) purifying the D-amino acid.
 66. A method ofpreventing or inhibiting infection by a virus in vivo, wherein themethod comprises administering to a subject in need thereof a virusmitogen in a sub-mitogenic amount sufficient to induce a protectiveimmune response against the virus in the subject.
 67. The method ofclaim 66, wherein the virus mitogen is administered to the subject inadmixture with a pharmaceutically acceptable carrier.
 68. The method ofclaim 66, wherein the virus mitogen is an animal or human virus mitogenin natural or recombinant form.
 69. A method of preventing or inhibitinginfection by a protozoan parasite in vivo, wherein the method comprisesadministering to a human in need thereof a protozoan parasite mitogen ina sub-mitogenic amount sufficient to induce a protective immune responseagainst the protozoan parasite in the human.
 70. The method of claim 69,wherein the protozoan parasite mitogen is administered to the human inadmixture with a pharmaceutically acceptable carrier.
 71. The method ofclaim 70, wherein the protozoan parasite mitogen is a mitogen ofPlasmodium berghei in natural or recombinant form.
 72. The method ofclaim 70, wherein the protozoan parasite mitogen is a mitogen orplasmodium falciparum of plasmodium vivax in natural or recombinantform.
 73. Any molecular modification of the gene or a fragment of thegene encoding for a racemase/mitogen that leads to the inhibition of theexpression of the protein by the parasite or virus (gene knock out), andfurther utilization of parasites or viruses lacking those activities invivo aiming at immunoprotective responses.
 74. Any molecularmodification of the gene or a fragment of the gene encoding for aracemase/mitogen that leads to the hyperexpression of the protein by theparasite or virus (gene transgenesis), and further utilization of theparasite or virus to produce high amounts of the protein aiming atproducing high amounts of the native protein.
 75. Any molecularmodification of the gene or a fragment of the gene encoding for aracemase/mitogen that leads to an attenuation of parasite or virusinfectivity, or interaction with a host cell, and further injection ofthe parasites or viruses in vivo aiming at immunoprotective responses.76. Any molecular modification (for instance directed mutagenesis) ofthe protein or of its active site that leads to the inhibition of itsenzymatic or its mitogenic activity and further injection of mutatedparasites or viruses in vivo aiming at immunoprotective responses. 77.Use of any molecular or biochemical modification of the enzymaticactivity of the racemase (inhibition of the active site) aiming atdeveloping specific immunotherapy.
 78. Any molecule or compound thatinhibits the enzymatic activity of the protein aiming at developing adrug against parasite or virus infection or specific treatment ofparasitic or viral disease.